Abstract:

The present invention relates generally to the field of molecular biology
and concerns a method for enhancing various economically important
characteristics in plants. More specifically, the present invention
concerns a method for improving yield-related traits, such as enhanced
yield and/or enhanced growth, or modified content of storage compounds in
plants by modulating expression in a plant of a nucleic acid encoding a
GRP (Growth Related Protein) polypeptide. The present invention also
concerns plants having modulated expression of a nucleic acid encoding a
GRP polypeptide, which plants have improved characterisitics relative to
control plants. The invention also provides hitherto unknown GRP-encoding
nucleic acids, and constructs comprising the same, useful in performing
the methods of the invention.

Claims:

1. Method for increasing seed yield of plants relative to control plants,
comprising modulating expression in a plant of a nucleic acid encoding an
AZ polypeptide, and optionally selecting for plants having increased
yield, wherein said AZ polypeptide comprises at least one ankyrin repeat
and at least one C3H1 Zinc finger domain.

2-154. (canceled)

Description:

[0001]The present invention relates generally to the field of molecular
biology and concerns a method for improving various plant characteristics
by modulating expression in a plant of a nucleic acid encoding a GRP
(Growth Related Protein). The present invention also concerns plants
having modulated expression of a nucleic acid encoding a GRP polypeptide,
which plants have improved characteristics relative to corresponding wild
type plants or other control plants. The invention also provides
constructs useful in the methods of the invention.

[0002]The ever-increasing world population and the dwindling supply of
arable land available for agriculture fuels research towards increasing
the efficiency of agriculture. Conventional means for crop and
horticultural improvements utilise selective breeding techniques to
identify plants having desirable characteristics. However, such selective
breeding techniques have several drawbacks, namely that these techniques
are typically labour intensive and result in plants that often contain
heterogeneous genetic components that may not always result in the
desirable trait being passed on from parent plants. Advances in molecular
biology have allowed mankind to modify the germplasm of animals and
plants. Genetic engineering of plants entails the isolation and
manipulation of genetic material (typically in the form of DNA or RNA)
and the subsequent introduction of that genetic material into a plant.
Such technology has the capacity to deliver crops or plants having
various improved economic, agronomic or horticultural traits.

[0003]A trait of particular economic interest is increased yield. Yield is
normally defined as the measurable produce of economic value from a crop.
This may be defined in terms of quantity and/or quality. Yield is
directly dependent on several factors, for example, the number and size
of the organs, plant architecture (for example, the number of branches),
seed production, leaf senescence and more. Root development, nutrient
uptake, stress tolerance and early vigour may also be important factors
in determining yield. Optimizing the above-mentioned factors may
therefore contribute to increasing crop yield.

[0004]Seed yield is a particularly important trait, since the seeds of
many plants are important for human and animal nutrition. Crops such as
corn, rice, wheat, canola and soybean account for over half the total
human caloric intake, whether through direct consumption of the seeds
themselves or through consumption of meat products raised on processed
seeds. They are also a source of sugars, oils and many kinds of
metabolites used in industrial processes. Seeds contain an embryo (the
source of new shoots and roots) and an endosperm (the source of nutrients
for embryo growth during germination and during early growth of
seedlings). The development of a seed involves many genes, and requires
the transfer of metabolites from the roots, leaves and stems into the
growing seed. The endosperm, in particular, assimilates the metabolic
precursors of carbohydrates, oils and proteins and synthesizes them into
storage macromolecules to fill out the grain.

[0005]Another important trait for many crops is early vigour. Improving
early vigour is an important objective of modern rice breeding programs
in both temperate and tropical rice cultivars. Long roots are important
for proper soil anchorage in water-seeded rice. Where rice is sown
directly into flooded fields, and where plants must emerge rapidly
through water, longer shoots are associated with vigour. Where
drill-seeding is practiced, longer mesocotyls and coleoptiles are
important for good seedling emergence. The ability to engineer early
vigour into plants would be of great importance in agriculture. For
example, poor early vigour has been a limitation to the introduction of
maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European
Atlantic.

[0006]A further important trait is that of improved abiotic stress
tolerance. Abiotic stress is a primary cause of crop loss worldwide,
reducing average yields for most major crop plants by more than 50% (Wang
et al., Planta (2003) 218: 1-14). Abiotic stresses may be caused by
drought, salinity, extremes of temperature, chemical toxicity and
oxidative stress. The ability to improve plant tolerance to abiotic
stress would be of great economic advantage to farmers worldwide and
would allow for the cultivation of crops during adverse conditions and in
territories where cultivation of crops may not otherwise be possible.

[0007]Crop yield may therefore be increased by optimising one of the
above-mentioned factors.

[0008]Depending on the end use, the modification of certain yield traits
may be favoured over others. For example for applications such as forage
or wood production, or bio-fuel resource, an increase in the vegetative
parts of a plant may be desirable, and for applications such as flour,
starch or oil production, an increase in seed parameters may be
particularly desirable. Even amongst the seed parameters, some may be
favoured over others, depending on the application. Various mechanisms
may contribute to increasing seed yield, whether that is in the form of
increased seed size or increased seed number.

[0009]One approach to increasing yield (seed yield and/or biomass) in
plants may be through modification of the inherent growth mechanisms of a
plant, such as the cell cycle or various signalling pathways involved in
plant growth or in defense mechanisms.

[0010]It has now been found that various characteristics may be improved
in plants by modulating expression in a plant of a nucleic acid encoding
a GRP polypeptide in a plant. The GRP polypeptide may be one of the
following: an Ankyrin-Zinc finger polypeptide (AZ), a SYT polypeptide; a
chloroplastic fructose-1,6-bisphosphatase (cpFBPase) polypeptide; a small
inducible kinase (SIK); a Class II homeodomain-leucine zipper (HD Zip)
transcription factor; and a SYB1 polypeptide. The improved
characteristics comprise yield related traits, such as enhanced yield
and/or enhanced growth, or modified content of storage compounds.

BACKGROUND

Ankyrin-Zinc Finger Polypeptide

[0011]Plant biomass is yield for forage crops like alfalfa, silage corn
and hay. Many proxies for yield have been used in grain crops. Chief
amongst these are estimates of plant size. Plant size can be measured in
many ways depending on species and developmental stage, but include total
plant dry weight, above-ground dry weight, above-ground fresh weight,
leaf area, stem volume, plant height, rosette diameter, leaf length, root
length, root mass, tiller number and leaf number. Many species maintain a
conservative ratio between the size of different parts of the plant at a
given developmental stage. These allometric relationships are used to
extrapolate from one of these measures of size to another (e.g. Tittonell
et al 2005 Agric Ecosys & Environ 105: 213). Plant size at an early
developmental stage will typically correlate with plant size later in
development. A larger plant with a greater leaf area can typically absorb
more light and carbon dioxide than a smaller plant and therefore will
likely gain a greater weight during the same period (Fasoula & Tollenaar
2005 Maydica 50:39). This is in addition to the potential continuation of
the micro-environmental or genetic advantage that the plant had to
achieve the larger size initially. There is a strong genetic component to
plant size and growth rate (e.g. ter Steege et al 2005 Plant Physiology
139:1078), and so for a range of diverse genotypes plant size under one
environmental condition is likely to correlate with size under another
(Hittalmani et al 2003 Theoretical Applied Genetics 107:679). In this way
a standard environment is used as a proxy for the diverse and dynamic
environments encountered at different locations and times by crops in the
field.

[0012]Harvest index, the ratio of seed yield to aboveground dry weight, is
relatively stable under many environmental conditions and so a robust
correlation between plant size and grain yield can often be obtained
(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are
intrinsically linked because the majority of grain biomass is dependent
on current or stored photosynthetic productivity by the leaves and stem
of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State
University Press, pp 68-73). Therefore, selecting for plant size, even at
early stages of development, has been used as an indicator for future
potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:
213). When testing for the impact of genetic differences on stress
tolerance, the ability to standardize soil properties, temperature, water
and nutrient availability and light intensity is an intrinsic advantage
of greenhouse or plant growth chamber environments compared to the field.
However, artificial limitations on yield due to poor pollination due to
the absence of wind or insects, or insufficient space for mature root or
canopy growth, can restrict the use of these controlled environments for
testing yield differences. Therefore, measurements of plant size in early
development, under standardized conditions in a growth chamber or
greenhouse, are standard practices to provide indication of potential
genetic yield advantages.

[0013]Transcription is performed by RNA polymerases. These polymerases are
usually associated with other proteins (transcription factors) that
determine the specificity of the transcription process. The transcription
factors bind to cis-regulatory elements of the gene and may also mediate
binding of other regulatory proteins. Stegmaier et al. proposed a
classification of transcription factors based on their DNA-binding
domains. 5 superclasses were discriminated, based on the presence of: 1)
basic domains, 2) zinc-coordinating domains, 3) helix-turn-helix domains,
4) beta scaffold domains with minor groove contacts and 0) other domains
(Stegmaier et al., Genome informatics 15, 276-286, 2004). The group of
transcription factors comprising zinc-coordinating domains is quite
divers and may be further classified according to their conserved
cysteine and histidine residues, including the WRKY domains, C6 zinc
clusters, DM and GCM domains.

[0014]Besides DNA binding motifs, transcription factors may also comprise
protein-protein interaction motifs. One such motif is the ankyrin motif.
It is present in very diverse families of proteins, usually as a repeat
of 2 to over 20 units. Each unit contains two antiparallel helices and a
beta-hairpin.

[0015]Although many plant proteins with zinc finger domains are well
characterised, little is known about plant proteins comprising the C3H1
zinc finger motif. PEI1, a transcription factor that reportedly plays a
role in embryo development, has a zinc finger motif that resembles the
C3H1 motif but lacks an ankyrin motif (Li and Thomas, Plant Cell 10,
383-398, 1998). WO 02/44389 describes AtSIZ, a transcription factor
isolated from Arabidopsis. Expression of AtSIZ under control of the
CaMV35S promoter was found to promote transcription of stress-induced
genes, and plants with increased expression of AtSIZ reportedly had a
higher survival rate under salt stress than control plants, but no
analysis was provided with respect to seed yield. It was postulated that
AtSIZ could be used for increasing resistance in plants to osmotic
stress.

SYT

[0016]Abiotic stresses such as drought stress, salinity stress, heat
stress and cold stress, or a combination of one or more of these, are
major limiting factors of plant growth and productivity (Boyer (1982)
Science 218: 443-448). These stresses have as common theme important for
plant growth water availability. Since high salt content in some soils
results in less available water for cell intake, its effect is similar to
those observed under drought conditions. Additionally, under freezing
temperatures, plant cells loose water as a result of ice formation that
starts from the apoplast and withdraws water from the symplast (McKersie
and Leshem (1994) Stress and stress coping in cultivated plants, Kluwer
Academic Publishers). During heat stress, stomata aperture is affected to
adjust cooling by evapotranspiration, thereby affecting the water content
of the plant. Commonly, a plant's molecular response mechanisms to each
of these stress conditions is similar.

[0017]Plants are exposed during their entire life cycle to conditions of
reduced environmental water content. Most plants have evolved strategies
to protect themselves against these conditions. However, if the severity
and duration of the stress conditions are too great, the effects on plant
development, growth and yield of most crop plant are profound. Continuous
exposure to reduced environmental water availability causes major
alterations in plant metabolism. These great changes in metabolism
ultimately lead to cell death and consequently to yield losses. Crop
losses and crop yield losses of major crops such as rice, maize (corn),
and wheat caused by these stresses represent a significant economic and
political factor and contribute to food shortages in many parts of the
world.

[0018]Another example of abiotic environmental stress is the reduced
availability of one or more nutrients that need to be assimilated by the
plants for growth and development. Because of the strong influence of
nutrition utilization efficiency on plant yield and product quality, a
huge amount of fertilizer is poured onto fields to optimize plant growth
and quality. Productivity of plants is limited by three primary
nutrients: phosphorous, potassium and nitrogen, which are usually the
rate-limiting elements in plant growth. The major nutritional element
required for plant growth is nitrogen (N). It is a constituent of
numerous important compounds found in living cells, including amino
acids, proteins (enzymes), nucleic acids, and chlorophyll. 1.5% to 2% of
plant dry matter is nitrogen and approximately 16% of total plant
protein. Thus, nitrogen availability is a major limiting factor in crop
plant growth and production (Frink et al. (1999) Proc Natl Acad Sci USA
96(4): 1175-1180), and has a major impact on protein accumulation and
amino acid composition. Therefore, of great interest are crop plants with
an increased yield when grown under nitrogen-limiting conditions.

[0019]Plant biomass is yield for forage crops like alfalfa, silage corn
and hay. Many proxies for yield have been used in grain crops. Chief
amongst these are estimates of plant size. Plant size can be measured in
many ways depending on species and developmental stage, but include total
plant dry weight, above-ground dry weight, above-ground fresh weight,
leaf area, stem volume, plant height, rosette diameter, leaf length, root
length, root mass, tiller number and leaf number. Many species maintain a
conservative ratio between the size of different parts of the plant at a
given developmental stage. These allometric relationships are used to
extrapolate from one of these measures of size to another (e.g. Tittonell
et al. (2005) Agric Ecosys & Environ 105: 213). Plant size at an early
developmental stage will typically correlate with plant size later in
development. A larger plant with a greater leaf area can typically absorb
more light and carbon dioxide than a smaller plant and therefore will
likely gain a greater weight during the same period (Fasoula & Tollenaar
(2005) Maydica 50:39). This is in addition to the potential continuation
of the micro-environmental or genetic advantage that the plant had to
achieve the larger size initially. There is a strong genetic component to
plant size and growth rate (e.g. ter Steege et al. (2005) Plant
Physiology 139:1078), and so for a range of diverse genotypes plant size
under one environmental condition is likely to correlate with size under
another (Hittalmani et al. (2003) Theoretical Applied Genetics 107:679).
In this way a standard environment is used as a proxy for the diverse and
dynamic environments encountered at different locations and times by
crops in the field.

[0020]Developing stress tolerant plants is a strategy that has the
potential to solve or mediate at least some aspects of yield loss
(McKersie and Leshem (1994) Stress and stress coping in cultivated
plants, Kluwer Academic Publishers). However, traditional breeding
strategies to develop new lines of plants that exhibit resistance
(tolerance) to these types of stresses are relatively slow and require
specific resistant lines for crossing with the desired line. Limited
germplasm resources for stress tolerance and incompatibility in crosses
between distantly related plant species represent significant problems
encountered in conventional breeding. Furthermore, such selective
breeding techniques are typically labour intensive and result in plants
that often contain heterogeneous genetic components that may not always
result in the desirable trait being passed on from parent plants.
Advances in molecular biology have allowed mankind to modify the
germplasm of animals and plants. Genetic engineering of plants entails
the isolation and manipulation of genetic material (typically in the form
of DNA or RNA) and the subsequent introduction of that genetic material
into a plant. Such technology has the capacity to deliver crops or plants
having various improved economic, agronomic or horticultural traits.

[0022]SYT belongs to a gene family of three members in Arabidopsis. The
SYT polypeptide shares homology with the human SYT. The human SYT
polypeptide was shown to be a transcriptional co-activator (Thaete et al.
(1999) Hum Molec Genet 8: 585-591). Three domains characterize the
mammalian SYT polypeptide: [0023](i) the N-terminal SNH (SYT N-terminal
homology) domain, conserved in mammals, plants, nematodes and fish;
[0024](ii) the C-terminal QPGY-rich domain, composed predominantly of
glycine, proline, glutamine and tyrosine, occurring at variable
intervals; [0025](iii) a methionine-rich (Met-rich) domain located
between the two previous domains.

[0026]In plant SYT polypeptides, the SNH domain is well conserved. The
C-terminal domain is rich in glycine and glutamine, but not in proline or
tyrosine. It has therefore been named the QG-rich domain in contrast to
the QPGY domain of mammals. As with mammalian SYT, a Met-rich domain may
be identified N-terminally of the QG domain. The QG-rich domain may be
taken to be substantially the C-terminal remainder of the polypeptide
(minus the SHN domain); the Met-rich domain is typically comprised within
the first half of the QG-rich (from the N-terminus to the C-terminus). A
second Met-rich domain may precede the SNH domain in plant SYT
polypeptides (see FIG. 1).

[0028]Overexpression of AN3 in Arabidopsis thaliana resulted in plants
with leaves that were 20-30% larger than those of the wild type
(Horiguchi et al. (2005) Plant J 43: 68-78).

[0029]In Japanese patent application 2004-350553, a method for controlling
the size of leaves in the horizontal direction is described, by
controlling the expression of the AN3 gene.

cpFBPase

[0030]Harvest index, the ratio of seed yield to aboveground dry weight, is
relatively stable under many environmental conditions and so a robust
correlation between plant size and grain yield can often be obtained
(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are
intrinsically linked because the majority of grain biomass is dependent
on current or stored photosynthetic productivity by the leaves and stem
of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State
University Press, pp 68-73). Therefore, selecting for plant size, even at
early stages of development, has been used as an indicator for future
potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:
213). When testing for the impact of genetic differences on stress
tolerance, the ability to standardize soil properties, temperature, water
and nutrient availability and light intensity is an intrinsic advantage
of greenhouse or plant growth chamber environments compared to the field.
However, artificial limitations on yield due to poor pollination due to
the absence of wind or insects, or insufficient space for mature root or
canopy growth, can restrict the use of these controlled environments for
testing yield differences. Therefore, measurements of plant size in early
development, under standardized conditions in a growth chamber or
greenhouse, are standard practices to provide indication of potential
genetic yield advantages.

[0031]Photosynthetic carbon metabolism in higher plants is an essential
process for plant growth and crop yield. Carbohydrates are produced in
higher plants by the fixation of atmospheric CO2 via the reductive
pentose phosphate (Calvin) pathway. This process takes place in the
chloroplast, and the newly synthesized triosephosphates can be kept in
the stromal compartment for starch synthesis, or may be exported to the
cytosol for sucrose formation. During photosynthesis, the newly
synthesized carbohydrate is channelled to one or the other form depending
on the needs of the plant and the environmental conditions.

[0032]The Calvin cycle is a complex pathway consisting of three phases of
thirteen reactions catalyzed by eleven enzymes. One of the more important
enzymes is chloroplastic fructose-1,6-bisphosphatase (cpFBPase), that
catalyzes the irreversible conversion of fructose-1,6-bisphosphate to
fructose-6-phosphate and Pi (inorganic P). The level of cpFBPase
polypeptide in the chloroplast is very low compared to those of the other
enzymes of the Calvin cycle.

[0033]The cpFBPase activity is regulated by the redox potential via the
ferredoxin/thioredoxin system, which modulates the enzyme activity in
response to light/dark conditions, and light-dependent changes in pH and
Mg2+ levels (Chiadmi et al. (1999) EMBO 18(23): 6809-6815). More
specifically, the cpFBPase polypeptide is active in the light, and
inactive in the dark, which takes place by a thioredoxin-mediated
reduction-oxidation interplay between SH groups of the enzyme molecule
and also via a light-induced rise in pH and Mg2+ concentration in the
chloroplast stroma (Buchanan (1980) Annu Rev Plant Physiol 31: 341-374;
Jacquot (1984) Bot Acta 103: 323-334).

[0034]Higher plant and algal cpFBPase polypeptides perform the same
enzymatic step, but differ somewhat in the regulation of enzymatic
activity. More specifically, algal cpFBPase polypeptides present a strict
requirement for reduction to be active, but are less strict on reductant
specificity, i.e., they can be activated by different plastidic
thioredoxins (Huppe and Buchanan (1989) Z Naturforsch 44(5-6): 487-94).

[0035]In photosynthetic (autotrophic) cells in addition to the cpFBPase
polypeptide, there is a second FBPase polypeptide (isoform) located in
the cytosol (cyFBPase) and involved in sucrose synthesis and
gluconeogenesis. The cyFBPase polypeptide presents very different
regulatory properties as compared to the cpFBPase polypeptide: it is
inhibited by excess substrate, displays an allosteric inhibition by AMP
and fructose-2,6-bisphophate, and presents a neutral pH optima. The
cyFBPase polypeptide is found in heterotrophic systems (such as animal
cells). An important difference between the cpFBPase polypeptides and the
cyFBPase polypeptides is the presence in the former of an amino acid
insertion that bears at least two conserved cysteine residues that are
the targets of thioredoxin regulation.

[0036]Transgenic potato plants expressing a potato cpFBPase nucleic acid
sequence under the control of a tuber-specific promoter were reported
(Thorbjornsen et al. (2002) Planta 214: 616-624). The authors observed
that the transgenic tubers did not differ from wild type tubers with
respect to starch content, or the levels of neutral sugars and
phosphorylated hexoses.

[0037]Two cyFBPase-encoding genes from cyanobacterium Synechococcus
(FBPase/SBPase and FBPasell) were operably linked to a tomato rbcS
transit peptide-coding sequence for chloroplastic subcellular targeting
of the chimeric proteins (Miyagawa et al. (2001) Nature Biotech 19:
965-969; Tamoi et al. (2006) Plant Cell Physiol 47(3): 380-390). The
activities of FBPase/SBPase and FBPasell polypeptides were not found to
be regulated by redox conditions via the ferredoxin/thioredoxin system,
since they lack the conserved cysteine residues. The tomato rbcS promoter
was used to control the expression of both chimeric genes. Transgenic
tobacco plants transformed with either chimeric construct were reported
to grow significantly faster and larger than wild type plants under
atmospheric conditions.

SIK

[0038]Plant breeders are often interested in improving specific aspects of
yield depending on the crop or plant in question, and the part of that
plant or crop which is of economic value. For example, for certain crops,
or for certain end uses, a plant breeder may look specifically for
improvements in plant biomass (weight) of one or more parts of a plant,
which may include aboveground (harvestable) parts and/or (harvestable)
parts below ground. This is particularly relevant where the aboveground
parts or below ground parts of a plant are for consumption. For many
crops, particularly cereals, it is an improvement in seed yield that is
highly desirable. Increased seed yield may manifest itself in many ways,
with each individual aspect of seed yield being of varying importance to
a plant breeder depending on the crop or plant in question and its end
use. For example, seed yield may be manifested as or result from a) an
increase in seed biomass (total seed weight) which may be on an
individual seed basis and/or per plant and/or per square meter; b)
increased number of flowers per plant; c) increased number of (filled)
seeds; d) increased seed filling rate (which is typically expressed as
the ratio between the number of filled seeds divided by the total number
of seeds; e) increased harvest index, which is typically expressed as a
ratio of the yield of harvestable parts, such as seeds, divided by the
total biomass; and f) increased thousand kernel weight (TKW), which is
typically extrapolated from the number of filled seeds counted and their
total weight.

[0039]An increase in seed yield may also be manifested as an increase in
seed size and/or seed volume. Furthermore, an increase in seed yield may
also manifest itself as an increase in seed area and/or seed length
and/or seed width and/or seed perimeter. Increased yield may also result
in modified architecture, or may occur because of modified architecture.

[0040]Taking corn as an example, a yield increase may be manifested as one
or more of the following: increase in the number of plants per square
meter, an increase in the number of ears per plant, an increase in the
number of rows, number of kernels per row, kernel weight, thousand kernel
weight, ear length/diameter, increase in the seed filling rate (which is
the number of filled seeds divided by the total number of seeds and
multiplied by 100), among others. Taking rice as an example, a yield
increase may manifest itself as an increase in one or more of the
following: number of plants per square meter, number of panicles per
plant, number of spikelets per panicle, number of flowers (florets) per
panicle (which is expressed as a ratio of the number of filled seeds over
the number of primary panicles), increase in the seed filling rate (which
is the number of filled seeds divided by the total number of seeds and
multiplied by 100), increase in thousand kernel weight, among others.

[0041]It would be of great advantage to a plant breeder to be able to be
able to pick and choose the aspects of seed yield to be altered. It would
be highly desirable to be able to pick off the shelf, so to speak, a gene
suitable for altering a particular aspect, or component, of seed yield.
For example an increase in the fill rate, combined with increased
thousand kernel weight would be highly desirable for a crop such as corn.
For rice and wheat a combination of increased fill rate, harvest index
and increased thousand kernel weight would be highly desirable.

[0042]Published International patent Application, WO 02/074801, in the
name of Genomine Inc., describes an AtSIK protein from Arabidopsis
thaliana and a gene encoding said protein. It is mentioned that plants
may be made resistant to osmotic stress by inhibiting expression of
AtSIK, and that as a consequence the productivity of a plant may be
increased. What is not mentioned however is which aspects of yield may be
modified by inhibiting expression of AtSIK.

HD Zip

[0043]The study and genetic manipulation of plants has a long history that
began even before the framed studies of Gregor Mendel. In perfecting this
science, scientists have accomplished modification of particular traits
in plants ranging from potato tubers having increased starch content to
oilseed plants such as canola and sunflower having increased or altered
fatty acid content. With the increased consumption and use of plant oils,
the modification of seed oil content and seed oil levels has become
increasingly widespread (e.g. Topfer et al., 1995, Science 268: 681-686).
Manipulation of biosynthetic pathways in transgenic plants provides a
number of opportunities for molecular biologists and plant biochemists to
affect plant metabolism giving rise to the production of specific
higher-value products. The seed oil production or composition has been
altered in numerous traditional oilseed plants such as soybean (U.S. Pat.
No. 5,955,650), canola (U.S. Pat. No. 5,955,650), sunflower (U.S. Pat.
No. 6,084,164), rapeseed (Topfer et al., 1995, Science 268:681-686), and
non-traditional oil seed plants such as tobacco (Cahoon et al., 1992,
Proc. Natl. Acad. Sci. USA89:11184-11188).

[0044]Plant seed oils comprise both neutral and polar lipids (See Table
1). The neutral lipids contain primarily triacylglycerol, which is the
main storage lipid that accumulates in oil bodies in seeds. The polar
lipids are mainly found in the various membranes of the seed cells, e.g.
the microsomal, plastidial, and mitochondrial membranes, and the cell
membrane. The neutral and polar lipids contain several common fatty acids
(See Table 2) and a range of less common fatty acids. The fatty acid
composition of membrane lipids is highly regulated and only a select
number of fatty acids are found in membrane lipids. On the other hand, a
large number of unusual fatty acids can be incorporated into the neutral
storage lipids in seeds of many plant species (Van de Loo F. J. et al.,
1993, Unusual Fatty Acids in Lipid Metabolism in Plants pp. 91-126,
editor T S Moore Jr. CRC Press; Millar et al., 2000, Trends Plant Sci.
5:95-101).

[0045]In Table 2, the fatty acids denoted with an asterisk do not normally
occur in plant seed oils, but their production in transgenic plant seed
oil is of importance in plant biotechnology.

[0046]The primary sites of fatty acid biosynthesis in plants are the
plastids. Fatty acid biosynthesis begins with the conversion of
acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase). The malonyl
moiety is then transferred to an acyl carrier protein (ACP) by the
malonyl-CoA:ACP transacylase. The enzyme beta-ketoacyl-ACP-synthase III
(KAS III) catalyzes the initial condensation reaction of fatty acid
biosynthesis, in which after decarboxylation of malonyl-ACP, the
resulting carbanion is transferred to acetyl-CoA by a nucleophilic attack
of the carbonyl-carbon, resulting in the formation of 3-ketobutyryl-ACP.
The reaction cycle is completed by a reduction, a dehydration and again a
reduction yielding butyric acid. This reaction cycle is repeated (with
KAS I or KAS II catalyzing the condensation reaction) until the
acyl-group reach a chain length of usually 16 to 18 carbon atoms. These
acyl-ACPs can be desaturated by the stearoyl-ACP desaturase, used as
substrates for plastidial acyltransferases in the formation of lipids
through what has been referred to as the prokaryotic pathway, or exported
to the cytosol after cleavage from ACP through the action of
thioesterases. In the cytosol they enter the acyl-CoA pool and can be
used for the synthesis of lipids through what has been referred to as the
eukaryotic pathway in the endoplasmic reticulum.

[0047]Lipid synthesis through both the prokaryotic and eukaryotic pathways
occurs through the successive acylation of glycerol-3-phosphate,
catalyzed by glycerol-3-phosphate acyltransferases (GPAT) and
lysophosphatidic acid acyltransferase (LPAAT) (Browse et al., 1986,
Biochemical J. 235:25-31; Ohlrogge & Browse, 1995, 5 Plant Ce11
7:957-970). The resulting phosphatidic acid (PA) is the precursor for
other polar membrane lipids such as monogalactosyldiacylglycerol (MGD),
digalactosyldiacylglycerol (DGD), phosphatidylglycerol (PG) and
sulfoquinovosyldiacylglycerol (SQD) in the plastid and
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI) and phosphatidylserine (PS) in the endoplasmic
reticulum. The polar lipids are also the sites of further modification of
the acyl-chain such as desaturation, acetylenation, and hydroxylation. In
the endoplasmic reticulum, PA is also the intermediate in the
biosynthesis of triacylglycerol (TAG), the major component of neutral
lipids and hence of seed oil. Furthermore, alternative pathways for the
biosynthesis of TAGS can exist (i.e. transacylation through the action of
phosphatidylcholine:diacylglycerol acyltransferase) (Voelker, 1996,
Genetic Engineering ed.:Setlow 1 8: 111-113; Shanklin & Cahoon, 1998,
Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641; Frentzen, 1998,
Lipids 100:161-166; Millar et al., 2000, Trends Plant Sci. 5:95-101). The
reverse reaction, the breakdown of triacylglycerol to diacylglycerol and
fatty acids is catalyzed by lipases. Such a breakdown can be seen towards
the end of seed development resulting in a certain reduction in seed oil.
(Buchanan et al., 2000).

[0048]Storage lipids in seeds are synthesized from carbohydrate-derived
precursors. Plants have a complete glycolytic pathway in the cytosol
(Plaxton, 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 185-214),
and it has been shown that a complete pathway also exists in the plastids
of rapeseed (Kang & Rawsthorne, 1994, Plant J. 6:795-805). Sucrose is the
primary source of carbon and energy, transported from the leaves into the
developing seeds. During the storage phase of seeds, sucrose is converted
in the cytosol to provide the metabolic precursors glucose-6-phosphate
and pyruvate. These are transported into the plastids and converted into
acetyl-CoA that serves as the primary precursor for the synthesis of
fatty acids. Acetyl-CoA in the plastids is the central precursor for
lipid biosynthesis. Acetyl-CoA can be formed in the plastids by different
reactions, and the exact contribution of each reaction is still being
debated (Ohlrogge & Browse, 1995, Plant Ce11 7:957-970). It is accepted,
however, that a large part of the acetyl-CoA is derived from
glucose-6-phospate and pyruvate that are imported from the cytoplasm into
the plastids. Sucrose is produced in the source organs (leaves, or
anywhere that photosynthesis occurs) and is transported to the developing
seeds, also termed sink organs. In the developing seeds, sucrose is the
precursor for all the storage compounds, i.e. starch, lipids, and partly
the seed storage proteins. Therefore, it is clear that carbohydrate
metabolism in which sucrose plays a central role is very important to the
accumulation of seed storage compounds.

[0049]Although lipid and fatty acid content of seed oil can be modified by
the traditional methods of plant breeding, the advent of recombinant DNA
technology has allowed for easier manipulation of the seed oil content of
a plant, and in some cases, has allowed for the alteration of seed oils
in ways that could not be accomplished by breeding alone (See, e.g.,
Topfer et al., 1995, Science 268:681-686). For example, introduction of a
Δ12-hydroxylase nucleic acid sequence into transgenic tobacco
resulted in the formation of a novel fatty acid, ricinoleic acid, into
the tobacco seed oil (Van de Loo et al., 1995, Proc. Natl. Acad. Sci USA
92:6743-6747). Tobacco plants have also been engineered to produce low
levels of petroselinic acid by the introduction and expression of an
acyl-ACP desaturase from coriander (Cahoon et al., 1992, Proc. Natl.
Acad. Sci USA 89: 11 184-11 188).

[0050]The modification of seed oil content in plants has significant
medical, nutritional, and economic ramifications. With regard to the
medical ramifications, the long chain fatty acids (C18 and longer) found
in many seed oils have been linked to reductions in hypercholesterolemia
and other clinical disorders related to coronary heart disease (Brenner,
1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a plant
having increased levels of these types of fatty acids may reduce the risk
of heart disease. Enhanced levels of seed oil content are also useful in
increasing the large-scale production of seed oils and thereby reducing
the cost of these oils.

[0051]As mentioned earlier, several desaturase nucleic acids such as the
Δ6-desaturase nucleic acid, Δ12-desaturase nucleic
acid and acyl-ACP desaturase nucleic acid have been cloned and
demonstrated to encode enzymes required for fatty acid synthesis in
various plant species. Oleosin nucleic acid sequences from species such
as Brassica, soybean, carrot, pine, and Arabidopsis thaliana have also
been cloned and determined to encode proteins associated with the
phospholipid monolayer membrane of oil bodies in those plants.

[0052]It has now been found that nucleic acid sequences encoding Class II
homeodomain-leucine zipper (HD-Zip) transcription factors are useful in
modifying the content of storage compounds in seeds.

[0053]Transcription factors are usually defined as proteins that show
sequence-specific DNA binding and that are capable of activating and/or
repressing transcription. The Arabidopsis genome codes for at least 1533
transcriptional regulators, which account for ˜5.9% of its
estimated total number of genes. About 45% of these transcription factors
are reported to be from families specific to plants (Riechmann et al.,
2000 (Science Vol. 290, 2105-2109)). One example of such a plant-specific
family of transcription factors is the family of HD-Zip transcription
factors.

[0054]Homeobox genes are transcription factors present in all eukaryotes
and constitute a gene family of at least 89 members in Arabidopsis
thaliana. They are characterized by the presence of a homeodomain, which
usually consists of 60 conserved amino acid residues that form a
helix-loop-helix-turn-helix structure that binds DNA. This DNA binding
site is usually pseudopalindromic. Homeobox genes play crucial and
diverse roles in many aspects of development, including the early
development of animal embryos, the specification of cell types in yeast,
and the initiation and maintenance of the shoot apical meristem in
flowering plants (Sakakibara et al. Mol. Biol. Evol. 18(4): 491-502,
2001).

[0055]Numerous angiosperm homeobox genes have been isolated and sorted
into seven distinct groups based on their amino acid similarities. These
include the KNOX, BELL, HD-PHD-finger, HAT1, HAT 2, GL2, and ATHB8
groups. Genes in the latter four groups also encode a leucine zipper
motif adjacent to the C-terminus of the homeodomain and collectively form
the homeodomain-leucine zipper (HD-Zip) gene family. Aso et al., Mol.
Biol. Evol. 16(4):544-552, 1999. Of the at least 89 members comprising
the homeobox gene family in Arabidopsis thaliana, at least 47 comprise
both a homeodomain and a leucine zipper. Although the combination of a
homeodomain and a leucine zipper motif is unique to plants, it has also
been encountered in moss in addition to vascular plants (Sakakibara et
al. (2001) Mol Biol Evol 18(4): 491-502).

[0056]Homeodomain leucine zipper (HD-Zip) proteins constitute a family of
transcription factors characterized by the presence of a DNA-binding
domain (HD) and an adjacent leucine zipper (Zip) motif. The leucine
zipper, adjacent to the C-terminal end of the homeodomain, consists of
several heptad repeats (at least four) in which a leucine (occasionally a
valine or an isoleucine) typically appears every seventh amino acid. The
leucine zipper is important for protein dimerisation. This dimerisation
is a prerequisite for DNA binding (Sessa et al. (1993) EMBO J 12(9):
3507-3517), and may proceed between two identical HD-Zip proteins
(homodimer) or between two different HD-Zip proteins (heterodimer).

[0057]The group of angiosperm homeobox genes HAT1, HAT2, GL2, and ATHB8
groups have been renamed the HD-Zip I-IV subfamilies. The combination of
a homeodomain and a leucine zipper motif is unique to higher plants,
suggesting that the HD-Zip genes may be involved in the regulation of
developmental processes specific to plants. Aso et al., Mol. Biol. Evol.
16(4):544-552, 1999. The functions of the HD-Zip genes are diverse among
the different subfamilies. HD-Zip I and II genes are likely involved in
signal transduction networks of light, dehydration-induced ABA, or auxin.
These signal transduction networks are related to the general growth
regulation of plants. The overexpression of sense or antisense HD-Zip I
or II mRNA usually alters growth rate and development. Most members of
the HD-Zip III subfamily play roles in cell differentiation in the stele.
HD-Zip IV genes are related to the differentiation of the outermost cell
layer. Sakakibara et al. Mol. Biol. Evol. 18(4): 491-502, 2001.

[0059]The Class II HD-Zip gene from Arabidopsis thaliana, HAT4, also known
as ATHB-2, has been reported to act as a regulator of shade-avoidance
responses (Morelli and Ruberti TIPS Vol. 7 No. 9, September 2002).

SYB1

[0060]Ran is a small signalling GTPase (GTP binding protein) in animal
cells, which is involved in nucleocytoplasmic transport.
Nucleocytoplasmic transport has been studied mainly in animal systems.
Several Ran binding proteins are known, among them RanBP2, also known as
Nup358. RanBP2 is postulated to associate on the cytoplasmic side with
proteins of the Nuclear Pore Complex and putatively functions as a SUMO
E3 ligase, although its structure is not of a typical E3 ligase. SUMOs
(small ubiquitin-related modifiers) are eukaryotic proteins that are
covalently ligated to other proteins, thereby regulating a number of
cellular processes. In association with Ubc9 (which acts an E2
conjugating protein), RanBP2 labels substrates linked to nuclear import
receptors with SUMO-1, in a similar way as ubiquitination of proteins.
The SUMOylated substrate is then imported through the nuclear pore into
the nucleus. Examples of imported proteins upon SUMOylation include
NFX1-p15, a chaperone in mRNA export and the histone deacetylase HDAC-4.
SUMO modification and hence RanBP2, is also implicated to play a role in
gene expression, in cell cycle (during nuclear envelope breakdown), and
in the formation of subcellular structures such as promyelocytic leukemia
bodies.

[0061]Most of the studies were performed in model animal systems and
little is known about the corresponding proteins and processes in plants.
Proteins related to RanBP2, identified in Arabidopsis, share the zinc
finger domains but are otherwise different in structure.

SUMMARY

[0062]Surprisingly, it has now been found that modulating expression of a
nucleic acid encoding a GRP polypeptide gives plants having improved
characteristics relative to control plants.

[0063]In one aspect of the present invention, it has now been found that
modulating expression in a plant of a nucleic acid encoding the AZ
polypeptide from Arabidopsis (AtAZ) or a homologue thereof gives plants
having increased yield relative to control plants. According to one
embodiment of the present invention, there is provided a method for
increasing plant yield, comprising modulating expression in a plant of a
nucleic acid encoding the AZ polypeptide or a homologue thereof.
Advantageously, performance of the methods according to the present
invention results in plants having increased yield, particularly seed
yield, relative to corresponding wild type plants. The present invention
also provides nucleic acid sequences and constructs useful in performing
such methods.

[0064]In another aspect of the present invention, it has now been found
that modulating expression in a plant of a nucleic acid sequence encoding
a SYT polypeptide gives plants having increased yield under abiotic
stress relative to control plants. Therefore, the invention also provides
a method for increasing plant yield under abiotic stress relative to
control plants, comprising modulating expression in a plant of a nucleic
acid sequence encoding a SYT polypeptide. The present invention also
provides nucleic acid sequences and constructs useful in performing such
methods.

[0065]In still another aspect of the present invention, it has now been
found that increasing expression in aboveground parts of a plant of a
nucleic acid sequence encoding a chloroplastic
fructose-1,6-bisphosphatase (cpFBPase) polypeptide increases plant yield
relative to control plants. Therefore, according to the present
invention, there is provided a method for increasing plant yield relative
to control plants, comprising increasing expression in aboveground parts
of a plant of a nucleic acid sequence encoding a cpFBPase polypeptide.
The present invention also provides nucleic acid sequences and constructs
useful in performing such methods.

[0066]In yet another aspect of the present invention, it has now been
found that modulating expression in a plant of a SIK nucleic acid and/or
a SIK polypeptide gives plants having various improved yield-related
traits relative to control plants, wherein overexpression of a
SIK-encoding nucleic acid in a plant gives increased number of flowers
per plant relative to control plants, and wherein the reduction or
substantial elimination of a SIK nucleic acid gives increased thousand
kernel weight, increased harvest index and increased fill rate relative
to corresponding wild type plants. The present invention also provides
nucleic acid sequences and constructs useful in performing such methods.

[0067]In a further aspect of the present invention, it has now been found
that nucleic acids encoding Class II HD-Zip transcription factors are
useful in modifying the content of storage compounds in seeds. The
present invention therefore provides a method for modifying the content
of storage compounds in seeds relative to control plants by modulating
expression in a plant of a nucleic acid encoding a Class II HD-Zip
transcription factor. The present invention also provides nucleic acid
sequences and constructs useful in performing such methods. The invention
further provides seeds having a modified content of storage compounds
relative to control plants, which seeds have modulated expression of a
nucleic acid encoding a Class II HD-Zip transcription factor. The present
invention provides a method for modifying the content of storage
compounds in seeds relative to control plants, comprising modulating
expression in a plant of a nucleic acid encoding a Class II HD-Zip
transcription factor.

[0068]In another aspect of the present invention, it has now been found
that modulating expression in a plant of a nucleic acid encoding a SYB1
polypeptide gives plants having enhanced yield-related traits relative to
control plants. This yield increase was surprisingly observed when the
plants were cultivated under conditions without stress (non-stress
conditions). The present invention therefore also provides a method for
enhancing yield-related traits in plants relative to control plants,
comprising modulating expression in a plant of a nucleic acid encoding a
SYB1 polypeptide. The present invention also provides nucleic acid
sequences and constructs useful in performing such methods.

DEFINITIONS

Polypeptide(s)/Protein(s)

[0069]The terms "polypeptide" and "protein" are used interchangeably
herein and refer to amino acids in a polymeric form of any length, linked
together by peptide bonds.

[0070]The terms "polynucleotide(s)", "nucleic acid sequence(s)",
"nucleotide sequence(s)", "nucleic acid(s)", "nucleic acid molecule" are
used interchangeably herein and refer to nucleotides, either
ribonucleotides or deoxyribonucleotides or a combination of both, in a
polymeric unbranched form of any length.

Control Plant(s)

[0071]The choice of suitable control plants is a routine part of an
experimental setup and may include corresponding wild type plants or
corresponding plants without the gene of interest. The control plant is
typically of the same plant species or even of the same variety as the
plant to be assessed. The control plant may also be a nullizygote of the
plant to be assessed. Nullizygotes are individuals missing the transgene
by segregation. A "control plant" as used herein refers not only to whole
plants, but also to plant parts, including seeds and seed parts.

[0072]Homoloque(s)

[0073]"Homologues" of a protein encompass peptides, oligopeptides,
polypeptides, proteins and enzymes having amino acid substitutions,
deletions and/or insertions relative to the unmodified protein in
question and having similar biological and functional activity as the
unmodified protein from which they are derived.

[0074]A deletion refers to removal of one or more amino acids from a
protein.

[0075]An insertion refers to one or more amino acid residues being
introduced into a predetermined site in a protein. Insertions may
comprise N-terminal and/or C-terminal fusions as well as intra-sequence
insertions of single or multiple amino acids. Generally, insertions
within the amino acid sequence will be smaller than N- or C-terminal
fusions, of the order of about 1 to 10 residues. Examples of N- or
C-terminal fusion proteins or peptides include the binding domain or
activation domain of a transcriptional activator as used in the yeast
two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione
S-transferase-tag, protein A, maltose-binding protein, dihydrofolate
reductase, TagΨ100 epitope, c-myc epitope, FLAG®-epitope, lacZ,
CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV
epitope.

[0076]A substitution refers to replacement of amino acids of the protein
with other amino acids having similar properties (such as similar
hydrophobicity, hydrophilicity, antigenicity, propensity to form or break
α-helical structures or β-sheet structures). Amino acid
substitutions are typically of single residues, but may be clustered
depending upon functional constraints placed upon the polypeptide;
insertions will usually be of the order of about 1 to 10 amino acid
residues. The amino acid substitutions are preferably conservative amino
acid substitutions. Conservative substitution tables are well known in
the art (see for example Creighton (1984) Proteins. W.H. Freeman and
Company (Eds) and Table 3 below).

[0077]Amino acid substitutions, deletions and/or insertions may readily be
made using peptide synthetic techniques well known in the art, such as
solid phase peptide synthesis and the like, or by recombinant DNA
manipulation. Methods for the manipulation of DNA sequences to produce
substitution, insertion or deletion variants of a protein are well known
in the art. For example, techniques for making substitution mutations at
predetermined sites in DNA are well known to those skilled in the art and
include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland,
Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego,
Calif.), PCR-mediated site-directed mutagenesis or other site-directed
mutagenesis protocols.

Derivatives

[0078]"Derivatives" include peptides, oligopeptides, polypeptides which
may, compared to the amino acid sequence of the naturally-occurring form
of the protein, such as the protein of interest, comprise substitutions
of amino acids with non-naturally occurring amino acid residues, or
additions of non-naturally occurring amino acid residues. "Derivatives"
of a protein also encompass peptides, oligopeptides, polypeptides which
comprise naturally occurring altered (glycosylated, acylated, prenylated,
phosphorylated, myristoylated, sulphated etc.) or non-naturally altered
amino acid residues compared to the amino acid sequence of a
naturally-occurring form of the polypeptide. A derivative may also
comprise one or more non-amino acid substituents or additions compared to
the amino acid sequence from which it is derived, for example a reporter
molecule or other ligand, covalently or non-covalently bound to the amino
acid sequence, such as a reporter molecule which is bound to facilitate
its detection, and non-naturally occurring amino acid residues relative
to the amino acid sequence of a naturally-occurring protein. Furthermore,
"derivatives" also include fusions of the naturally-occurring form of the
protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a
review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60,
523-533, 2003).

Ortholoque(s)/Paraloque(s)

[0079]Orthologues and paralogues encompass evolutionary concepts used to
describe the ancestral relationships of genes. Paralogues are genes
within the same species that have originated through duplication of an
ancestral gene; orthologues are genes from different organisms that have
originated through speciation, and are also derived from a common
ancestral gene.

Domain

[0080]The term "domain" refers to a set of amino acids conserved at
specific positions along an alignment of sequences of evolutionarily
related proteins. While amino acids at other positions can vary between
homologues, amino acids that are highly conserved at specific positions
indicate amino acids that are likely essential in the structure,
stability or function of a protein. Identified by their high degree of
conservation in aligned sequences of a family of protein homologues, they
can be used as identifiers to determine if any polypeptide in question
belongs to a previously identified polypeptide family.

Motif/Consensus Sequence/Signature

[0081]The term "motif" or "consensus sequence" or "signature" refers to a
short conserved region in the sequence of evolutionarily related
proteins. Motifs are frequently highly conserved parts of domains, but
may also include only part of the domain, or be located outside of
conserved domain (if all of the amino acids of the motif fall outside of
a defined domain).

Hybridisation

[0082]The term "hybridisation" as defined herein is a process wherein
substantially homologous complementary nucleotide sequences anneal to
each other. The hybridisation process can occur entirely in solution,
i.e. both complementary nucleic acids are in solution. The hybridisation
process can also occur with one of the complementary nucleic acids
immobilised to a matrix such as magnetic beads, Sepharose beads or any
other resin. The hybridisation process can furthermore occur with one of
the complementary nucleic acids immobilised to a solid support such as a
nitro-cellulose or nylon membrane or immobilised by e.g. photolithography
to, for example, a siliceous glass support (the latter known as nucleic
acid arrays or microarrays or as nucleic acid chips). In order to allow
hybridisation to occur, the nucleic acid molecules are generally
thermally or chemically denatured to melt a double strand into two single
strands and/or to remove hairpins or other secondary structures from
single stranded nucleic acids.

[0083]The term "stringency" refers to the conditions under which a
hybridisation takes place. The stringency of hybridisation is influenced
by conditions such as temperature, salt concentration, ionic strength and
hybridisation buffer composition. Generally, low stringency conditions
are selected to be about 30° C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength and
pH. Medium stringency conditions are when the temperature is 20°
C. below Tm, and high stringency conditions are when the temperature
is 10° C. below Tm. High stringency hybridisation conditions
are typically used for isolating hybridising sequences that have high
sequence similarity to the target nucleic acid sequence. However, nucleic
acids may deviate in sequence and still encode a substantially identical
polypeptide, due to the degeneracy of the genetic code. Therefore medium
stringency hybridisation conditions may sometimes be needed to identify
such nucleic acid molecules.

[0084]The Tm is the temperature under defined ionic strength and pH, at
which 50% of the target sequence hybridises to a perfectly matched probe.
The Tm is dependent upon the solution conditions and the base
composition and length of the probe. For example, longer sequences
hybridise specifically at higher temperatures. The maximum rate of
hybridisation is obtained from about 16° C. up to 32° C.
below Tm. The presence of monovalent cations in the hybridisation
solution reduce the electrostatic repulsion between the two nucleic acid
strands thereby promoting hybrid formation; this effect is visible for
sodium concentrations of up to 0.4 M (for higher concentrations, this
effect may be ignored). Formamide reduces the melting temperature of
DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent
formamide, and addition of 50% formamide allows hybridisation to be
performed at 30 to 45° C., though the rate of hybridisation will
be lowered. Base pair mismatches reduce the hybridisation rate and the
thermal stability of the duplexes. On average and for large probes, the
Tm decreases about 1° C. per % base mismatch. The Tm may be
calculated using the following equations, depending on the types of
hybrids:

3) oligo-DNA or oligo-RNAd hybrids: [0086]For <20 nucleotides:
Tm=2 (In) [0087]For 20-35 nucleotides: Tm=22+1.46
(In) [0088]a or for other monovalent cation, but only accurate
in the 0.01-0.4 M range. [0089]b only accurate for % GC in the 30%
to 75% range. [0090]cL=length of duplex in base pairs. [0091]d
oligo, oligonucleotide; In,=effective length of primer=2×(no.
of G/C)+(no. of NT).

[0092]Non-specific binding may be controlled using any one of a number of
known techniques such as, for example, blocking the membrane with protein
containing solutions, additions of heterologous RNA, DNA, and SDS to the
hybridisation buffer, and treatment with Rnase. For non-homologous
probes, a series of hybridizations may be performed by varying one of (i)
progressively lowering the annealing temperature (for example from
68° C. to 42° C.) or (ii) progressively lowering the
formamide concentration (for example from 50% to 0%). The skilled artisan
is aware of various parameters which may be altered during hybridisation
and which will either maintain or change the stringency conditions.

[0093]Besides the hybridisation conditions, specificity of hybridisation
typically also depends on the function of post-hybridisation washes. To
remove background resulting from non-specific hybridisation, samples are
washed with dilute salt solutions. Critical factors of such washes
include the ionic strength and temperature of the final wash solution:
the lower the salt concentration and the higher the wash temperature, the
higher the stringency of the wash.

[0094]Wash conditions are typically performed at or below hybridisation
stringency. A positive hybridisation gives a signal that is at least
twice of that of the background. Generally, suitable stringent conditions
for nucleic acid hybridisation assays or gene amplification detection
procedures are as set forth above. More or less stringent conditions may
also be selected. The skilled artisan is aware of various parameters
which may be altered during washing and which will either maintain or
change the stringency conditions.

[0095]For example, typical high stringency hybridisation conditions for
DNA hybrids longer than 50 nucleotides encompass hybridisation at
65° C. in 1×SSC or at 42° C. in 1×SSC and 50%
formamide, followed by washing at 65° C. in 0.3×SSC.
Examples of medium stringency hybridisation conditions for DNA hybrids
longer than 50 nucleotides encompass hybridisation at 50° C. in
4×SSC or at 40° C. in 6×SSC and 50% formamide,
followed by washing at 50° C. in 2×SSC. The length of the
hybrid is the anticipated length for the hybridising nucleic acid. When
nucleic acids of known sequence are hybridised, the hybrid length may be
determined by aligning the sequences and identifying the conserved
regions described herein. 1×SSC is 0.15 M NaCl and 15 mM sodium
citrate; the hybridisation solution and wash solutions may additionally
include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured,
fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.

[0096]For the purposes of defining the level of stringency, reference can
be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,
3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or
to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989
and yearly updates).

Splice Variant

[0097]The term "splice variant" as used herein encompasses variants of a
nucleic acid sequence in which selected introns and/or exons have been
excised, replaced, displaced or added, or in which introns have been
shortened or lengthened. Such variants will be ones in which the
biological activity of the protein is substantially retained; this may be
achieved by selectively retaining functional segments of the protein.
Such splice variants may be found in nature or may be manmade. Methods
for predicting and isolating such splice variants are well known in the
art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).

Allelic Variant

[0098]Alleles or allelic variants are alternative forms of a given gene,
located at the same chromosomal position. Allelic variants encompass
Single Nucleotide Polymorphisms (SNPs), as well as Small
Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually
less than 100 bp. SNPs and INDELs form the largest set of sequence
variants in naturally occurring polymorphic strains of most organisms.

[0100]The terms "regulatory element", "control sequence" and "promoter"
are all used interchangeably herein and are to be taken in a broad
context to refer to regulatory nucleic acid sequences capable of
effecting expression of the sequences to which they are ligated. The term
"promoter" typically refers to a nucleic acid control sequence located
upstream from the transcriptional start of a gene and which is involved
in recognising and binding of RNA polymerase and other proteins, thereby
directing transcription of an operably linked nucleic acid. Encompassed
by the aforementioned terms are transcriptional regulatory sequences
derived from a classical eukaryotic genomic gene (including the TATA box
which is required for accurate transcription initiation, with or without
a CCAAT box sequence) and additional regulatory elements (i.e. upstream
activating sequences, enhancers and silencers) which alter gene
expression in response to developmental and/or external stimuli, or in a
tissue-specific manner. Also included within the term is a
transcriptional regulatory sequence of a classical prokaryotic gene, in
which case it may include a -35 box sequence and/or -10 box
transcriptional regulatory sequences. The term "regulatory element" also
encompasses a synthetic fusion molecule or derivative that confers,
activates or enhances expression of a nucleic acid molecule in a cell,
tissue or organ.

[0101]A "plant promoter" comprises regulatory elements, which mediate the
expression of a coding sequence segment in plant cells. Accordingly, a
plant promoter need not be of plant origin, but may originate from
viruses or micro-organisms, for example from viruses which attack plant
cells. The "plant promoter" can also originate from a plant cell, e.g.
from the plant which is transformed with the nucleic acid sequence to be
expressed in the inventive process and described herein. This also
applies to other "plant" regulatory signals, such as "plant" terminators.
The promoters upstream of the nucleotide sequences useful in the methods
of the present invention can be modified by one or more nucleotide
substitution(s), insertion(s) and/or deletion(s) without interfering with
the functionality or activity of either the promoters, the open reading
frame (ORF) or the 3'-regulatory region such as terminators or other 3'
regulatory regions which are located away from the ORF. It is furthermore
possible that the activity of the promoters is increased by modification
of their sequence, or that they are replaced completely by more active
promoters, even promoters from heterologous organisms. For expression in
plants, the nucleic acid molecule must, as described above, be linked
operably to or comprise a suitable promoter which expresses the gene at
the right point in time and with the required spatial expression pattern.

[0102]For the identification of functionally equivalent promoters, the
promoter strength and/or expression pattern of a candidate promoter may
be analysed for example by operably linking the promoter to a reporter
gene and assaying the expression level and pattern of the reporter gene
in various tissues of the plant. Suitable well-known reporter genes
include for example beta-glucuronidase or beta-galactosidase. The
promoter activity is assayed by measuring the enzymatic activity of the
beta-glucuronidase or beta-galactosidase. The promoter strength and/or
expression pattern may then be compared to that of a reference promoter
(such as the one used in the methods of the present invention).
Alternatively, promoter strength may be assayed by quantifying mRNA
levels or by comparing mRNA levels of the nucleic acid used in the
methods of the present invention, with mRNA levels of housekeeping genes
such as 18S rRNA, using methods known in the art, such as Northern
blotting with densitometric analysis of autoradiograms, quantitative
real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).
Generally by "weak promoter" is intended a promoter that drives
expression of a coding sequence at a low level. By "low level" is
intended at levels of about 1/10,000 transcripts to about 1/100,000
transcripts, to about 1/500,0000 transcripts per cell. Conversely, a
"strong promoter" drives expression of a coding sequence at high level,
or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000
transcripts per cell.

Operably Linked

[0103]The term "operably linked" as used herein refers to a functional
linkage between the promoter sequence and the gene of interest, such that
the promoter sequence is able to initiate transcription of the gene of
interest.

Constitutive Promoter

[0104]A "constitutive promoter" refers to a promoter that is
transcriptionally active during most, but not necessarily all, phases of
growth and development and under most environmental conditions, in at
least one cell, tissue or organ. Table 4a below gives examples of
constitutive promoters.

[0105]A ubiquitous promoter is active in substantially all tissues or
cells of an organism.

Developmentally-Regulated Promoter

[0106]A developmentally-regulated promoter is active during certain
developmental stages or in parts of the plant that undergo developmental
changes.

Inducible Promoter

[0107]An inducible promoter has induced or increased transcription
initiation in response to a chemical (for a review see Gatz 1997, Annu.
Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental or
physical stimulus, or may be "stress-inducible", i.e. activated when a
plant is exposed to various stress conditions, or a "pathogen-inducible"
i.e. activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

[0108]An organ-specific or tissue-specific promoter is one that is capable
of preferentially initiating transcription in certain organs or tissues,
such as the leaves, roots, seed tissue etc. For example, a "root-specific
promoter" is a promoter that is transcriptionally active predominantly in
plant roots, substantially to the exclusion of any other parts of a
plant, whilst still allowing for any leaky expression in these other
plant parts. Promoters able to initiate transcription in certain cells
only are referred to herein as "cell-specific".

[0109]A seed-specific promoter is transcriptionally active predominantly
in seed tissue, but not necessarily exclusively in seed tissue (in cases
of leaky expression). The seed-specific promoter may be active during
seed development and/or during germination. The seed specific promoter
may be endosperm/aleurone/embryo specific. Examples of seed-specific
promoters are shown in Tables 4b to 4e below. Further examples of
seed-specific promoters are given in Qing Qu and Takaiwa (Plant
Biotechnol. J. 2, 113-125, 2004), which disclosure is incorporated by
reference herein as if fully set forth.

[0110]A green tissue-specific promoter as defined herein is a promoter
that is transcriptionally active predominantly in green tissue,
substantially to the exclusion of any other parts of a plant, whilst
still allowing for any leaky expression in these other plant parts.

[0111]Another example of a tissue-specific promoter is a meristem-specific
promoter, which is transcriptionally active predominantly in meristematic
tissue, substantially to the exclusion of any other parts of a plant,
whilst still allowing for any leaky expression in these other plant
parts.

Terminator

[0112]The term "terminator" encompasses a control sequence which is a DNA
sequence at the end of a transcriptional unit which signals 3' processing
and polyadenylation of a primary transcript and termination of
transcription. The terminator can be derived from the natural gene, from
a variety of other plant genes, or from T-DNA. The terminator to be added
may be derived from, for example, the nopaline synthase or octopine
synthase genes, or alternatively from another plant gene, or less
preferably from any other eukaryotic gene.

Modulation

[0113]The term "modulation" means in relation to expression or gene
expression, a process in which the expression level is changed by said
gene expression in comparison to the control plant, the expression level
may be increased or decreased. The original, unmodulated expression may
be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA
with subsequent translation. The term "modulating the activity" shall
mean any change of the expression of the inventive nucleic acid sequences
or encoded proteins, which leads to increased yield and/or increased
growth of the plants.

Expression

[0114]The term "expression" or "gene expression" means the transcription
of a specific gene or specific genes or specific genetic construct. The
term "expression" or "gene expression" in particular means the
transcription of a gene or genes or genetic construct into structural RNA
(rRNA, tRNA) or mRNA with or without subsequent translation of the latter
into a protein. The process includes transcription of DNA and processing
of the resulting mRNA product.

Increased Expression/Overexpression

[0115]The term "increased expression" or "overexpression" as used herein
means any form of expression that is additional to the original wild-type
expression level. Methods for increasing expression of genes or gene
products are well documented in the art and include, for example,
overexpression driven by appropriate promoters, the use of transcription
enhancers or translation enhancers. Isolated nucleic acids which serve as
promoter or enhancer elements may be introduced in an appropriate
position (typically upstream) of a non-heterologous form of a
polynucleotide so as to upregulate expression of a nucleic acid encoding
the polypeptide of interest. For example, endogenous promoters may be
altered in vivo by mutation, deletion, and/or substitution (see, Kmiec,
U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated
promoters may be introduced into a plant cell in the proper orientation
and distance from a gene of the present invention so as to control the
expression of the gene.

[0116]If polypeptide expression is desired, it is generally desirable to
include a polyadenylation region at the 3'-end of a polynucleotide coding
region. The polyadenylation region can be derived from the natural gene,
from a variety of other plant genes, or from T-DNA. The 3' end sequence
to be added may be derived from, for example, the nopaline synthase or
octopine synthase genes, or alternatively from another plant gene, or
less preferably from any other eukaryotic gene.

[0117]An intron sequence may also be added to the 5' untranslated region
(UTR) or the coding sequence of the partial coding sequence to increase
the amount of the mature message that accumulates in the cytosol.
Inclusion of a spliceable intron in the transcription unit in both plant
and animal expression constructs has been shown to increase gene
expression at both the mRNA and protein levels up to 1000-fold (Buchman
and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes
Dev 1:1183-1200). Such intron enhancement of gene expression is typically
greatest when placed near the 5' end of the transcription unit. Use of
the maize introns Adh1-5 intron 1, 2, and 6, the Bronze-1 intron are
known in the art. For general information see: The Maize Handbook,
Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Endogenous Gene

[0118]Reference herein to an "endogenous" gene not only refers to the gene
in question as found in a plant in its natural form (i.e., without there
being any human intervention), but also refers to that same gene (or a
substantially homologous nucleic acid/gene) in an isolated form
subsequently (re)introduced into a plant (a transgene). For example, a
transgenic plant containing such a transgene may encounter a substantial
reduction of the transgene expression and/or substantial reduction of
expression of the endogenous gene. The isolated gene may be isolated from
an organism or may be manmade, for example by chemical synthesis.

Decreased Expression

[0119]Reference herein to "decreased epression" or "reduction or
substantial elimination" of expression is taken to mean a decrease in
endogenous gene expression and/or polypeptide levels and/or polypeptide
activity relative to control plants. The reduction or substantial
elimination is in increasing order of preference at least 10%, 20%, 30%,
40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more
reduced compared to that of control plants.

[0120]For the reduction or substantial elimination of expression an
endogenous gene in a plant, a sufficient length of substantially
contiguous nucleotides of a nucleic acid sequence is required. In order
to perform gene silencing, this may be as little as 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as
much as the entire gene (including the 5' and/or 3' UTR, either in part
or in whole). The stretch of substantially contiguous nucleotides may be
derived from the nucleic acid encoding the protein of interest (target
gene), or from any nucleic acid capable of encoding an orthologue,
paralogue or homologue of the protein of interest. Preferably, the
stretch of substantially contiguous nucleotides is capable of forming
hydrogen bonds with the target gene (either sense or antisense strand),
more preferably, the stretch of substantially contiguous nucleotides has,
in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either
sense or antisense strand). A nucleic acid sequence encoding a
(functional) polypeptide is not a requirement for the various methods
discussed herein for the reduction or substantial elimination of
expression of an endogenous gene.

[0121]This reduction or substantial elimination of expression may be
achieved using routine tools and techniques. A preferred method for the
reduction or substantial elimination of endogenous gene expression is by
introducing and expressing in a plant a genetic construct into which the
nucleic acid (in this case a stretch of substantially contiguous
nucleotides derived from the gene of interest, or from any nucleic acid
capable of encoding an orthologue, paralogue or homologue of any one of
the protein of interest) is cloned as an inverted repeat (in part or
completely), separated by a spacer (non-coding DNA).

[0122]In such a preferred method, expression of the endogenous gene is
reduced or substantially eliminated through RNA-mediated silencing using
an inverted repeat of a nucleic acid or a part thereof (in this case a
stretch of substantially contiguous nucleotides derived from the gene of
interest, or from any nucleic acid capable of encoding an orthologue,
paralogue or homologue of the protein of interest), preferably capable of
forming a hairpin structure. The inverted repeat is cloned in an
expression vector comprising control sequences. A non-coding DNA nucleic
acid sequence (a spacer, for example a matrix attachment region fragment
(MAR), an intron, a polylinker, etc.) is located between the two inverted
nucleic acids forming the inverted repeat. After transcription of the
inverted repeat, a chimeric RNA with a self-complementary structure is
formed (partial or complete). This double-stranded RNA structure is
referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the
plant into siRNAs that are incorporated into an RNA-induced silencing
complex (RISC). The RISC further cleaves the mRNA transcripts, thereby
substantially reducing the number of mRNA transcripts to be translated
into polypeptides. For further general details see for example, Grierson
et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).

[0123]Performance of the methods of the invention does not rely on
introducing and expressing in a plant a genetic construct into which the
nucleic acid is cloned as an inverted repeat, but any one or more of
several well-known "gene silencing" methods may be used to achieve the
same effects.

[0124]One such method for the reduction of endogenous gene expression is
RNA-mediated silencing of gene expression (downregulation). Silencing in
this case is triggered in a plant by a double stranded RNA sequence
(dsRNA) that is substantially similar to the target endogenous gene. This
dsRNA is further processed by the plant into about 20 to about 26
nucleotides called short interfering RNAs (siRNAs). The siRNAs are
incorporated into an RNA-induced silencing complex (RISC) that cleaves
the mRNA transcript of the endogenous target gene, thereby substantially
reducing the number of mRNA transcripts to be translated into a
polypeptide. Preferably, the double stranded RNA sequence corresponds to
a target gene.

[0125]Another example of an RNA silencing method involves the introduction
of nucleic acid sequences or parts thereof (in this case a stretch of
substantially contiguous nucleotides derived from the gene of interest,
or from any nucleic acid capable of encoding an orthologue, paralogue or
homologue of the protein of interest) in a sense orientation into a
plant. "Sense orientation" refers to a DNA sequence that is homologous to
an mRNA transcript thereof. Introduced into a plant would therefore be at
least one copy of the nucleic acid sequence. The additional nucleic acid
sequence will reduce expression of the endogenous gene, giving rise to a
phenomenon known as co-suppression. The reduction of gene expression will
be more pronounced if several additional copies of a nucleic acid
sequence are introduced into the plant, as there is a positive
correlation between high transcript levels and the triggering of
co-suppression.

[0126]Another example of an RNA silencing method involves the use of
antisense nucleic acid sequences. An "antisense" nucleic acid sequence
comprises a nucleotide sequence that is complementary to a "sense"
nucleic acid sequence encoding a protein, i.e. complementary to the
coding strand of a double-stranded cDNA molecule or complementary to an
mRNA transcript sequence. The antisense nucleic acid sequence is
preferably complementary to the endogenous gene to be silenced. The
complementarity may be located in the "coding region" and/or in the
"non-coding region" of a gene. The term "coding region" refers to a
region of the nucleotide sequence comprising codons that are translated
into amino acid residues. The term "non-coding region" refers to 5' and
3' sequences that flank the coding region that are transcribed but not
translated into amino acids (also referred to as 5' and 3' untranslated
regions).

[0127]Antisense nucleic acid sequences can be designed according to the
rules of Watson and Crick base pairing. The antisense nucleic acid
sequence may be complementary to the entire nucleic acid sequence (in
this case a stretch of substantially contiguous nucleotides derived from
the gene of interest, or from any nucleic acid capable of encoding an
orthologue, paralogue or homologue of the protein of interest), but may
also be an oligonucleotide that is antisense to only a part of the
nucleic acid sequence (including the mRNA 5' and 3' UTR). For example,
the antisense oligonucleotide sequence may be complementary to the region
surrounding the translation start site of an mRNA transcript encoding a
polypeptide. The length of a suitable antisense oligonucleotide sequence
is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20,
15 or 10 nucleotides in length or less. An antisense nucleic acid
sequence according to the invention may be constructed using chemical
synthesis and enzymatic ligation reactions using methods known in the
art. For example, an antisense nucleic acid sequence (e.g., an antisense
oligonucleotide sequence) may be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase the
physical stability of the duplex formed between the antisense and sense
nucleic acid sequences, e.g., phosphorothioate derivatives and acridine
substituted nucleotides may be used. Examples of modified nucleotides
that may be used to generate the antisense nucleic acid sequences are
well known in the art. Known nucleotide modifications include
methylation, cyclization and `caps` and substitution of one or more of
the naturally occurring nucleotides with an analogue such as inosine.
Other modifications of nucleotides are well known in the art.

[0128]The antisense nucleic acid sequence can be produced biologically
using an expression vector into which a nucleic acid sequence has been
subcloned in an antisense orientation (i.e., RNA transcribed from the
inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of interest). Preferably, production of antisense nucleic
acid sequences in plants occurs by means of a stably integrated nucleic
acid construct comprising a promoter, an operably linked antisense
oligonucleotide, and a terminator.

[0129]The nucleic acid molecules used for silencing in the methods of the
invention (whether introduced into a plant or generated in situ)
hybridize with or bind to mRNA transcripts and/or genomic DNA encoding a
polypeptide to thereby inhibit expression of the protein, e.g., by
inhibiting transcription and/or translation. The hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the case of an antisense nucleic acid sequence which binds to
DNA duplexes, through specific interactions in the major groove of the
double helix. Antisense nucleic acid sequences may be introduced into a
plant by transformation or direct injection at a specific tissue site.
Alternatively, antisense nucleic acid sequences can be modified to target
selected cells and then administered systemically. For example, for
systemic administration, antisense nucleic acid sequences can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid
sequence to peptides or antibodies which bind to cell surface receptors
or antigens. The antisense nucleic acid sequences can also be delivered
to cells using the vectors described herein.

[0131]The reduction or substantial elimination of endogenous gene
expression may also be performed using ribozymes. Ribozymes are catalytic
RNA molecules with ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid sequence, such as an mRNA, to which they
have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be
used to catalytically cleave mRNA transcripts encoding a polypeptide,
thereby substantially reducing the number of mRNA transcripts to be
translated into a polypeptide. A ribozyme having specificity for a
nucleic acid sequence can be designed (see for example: Cech et al. U.S.
Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
Alternatively, mRNA transcripts corresponding to a nucleic acid sequence
can be used to select a catalytic RNA having a specific ribonuclease
activity from a pool of RNA molecules (Bartel and Szostak (1993) Science
261, 1411-1418). The use of ribozymes for gene silencing in plants is
known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.
(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.
(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

[0132]Gene silencing may also be achieved by insertion mutagenesis (for
example, T-DNA insertion or transposon insertion) or by strategies as
described by, among others, Angell and Baulcombe ((1999) Plant J 20(3):
357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

[0133]Gene silencing may also occur if there is a mutation on an
endogenous gene and/or a mutation on an isolated gene/nucleic acid
subsequently introduced into a plant. The reduction or substantial
elimination may be caused by a non-functional polypeptide. For example,
the polypeptide may bind to various interacting proteins; one or more
mutation(s) and/or truncation(s) may therefore provide for a polypeptide
that is still able to bind interacting proteins (such as receptor
proteins) but that cannot exhibit its normal function (such as signalling
ligand).

[0135]Other methods, such as the use of antibodies directed to an
endogenous polypeptide for inhibiting its function in planta, or
interference in the signalling pathway in which a polypeptide is
involved, will be well known to the skilled man. In particular, it can be
envisaged that manmade molecules may be useful for inhibiting the
biological function of a target polypeptide, or for interfering with the
signalling pathway in which the target polypeptide is involved.

[0136]Alternatively, a screening program may be set up to identify in a
plant population natural variants of a gene, which variants encode
polypeptides with reduced activity. Such natural variants may also be
used for example, to perform homologous recombination. Artificial and/or
natural microRNAs (miRNAs) may be used to knock out gene expression
and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs
of typically 19-24 nucleotides long. They function primarily to regulate
gene expression and/or mRNA translation. Most plant microRNAs (miRNAs)
have perfect or near-perfect complementarity with their target sequences.
However, there are natural targets with up to five mismatches. They are
processed from longer non-coding RNAs with characteristic fold-back
structures by double-strand specific RNases of the Dicer family. Upon
processing, they are incorporated in the RNA-induced silencing complex
(RISC) by binding to its main component, an Argonaute protein. MiRNAs
serve as the specificity components of RISC, since they base-pair to
target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent
regulatory events include target mRNA cleavage and destruction and/or
translational inhibition. Effects of miRNA overexpression are thus often
reflected in decreased mRNA levels of target genes.

[0137]Artificial microRNAs (amiRNAs), which are typically 21 nucleotides
in length, can be genetically engineered specifically to negatively
regulate gene expression of single or multiple genes of interest.
Determinants of plant microRNA target selection are well known in the
art. Empirical parameters for target recognition have been defined and
can be used to aid in the design of specific amiRNAs, (Schwab et al.,
Dev. Cell 8, 517-527, 2005). Convenient tools for design and generation
of amiRNAs and their precursors are also available to the public (Schwab
et al., Plant Cell 18, 1121-1133, 2006).

[0138]For optimal performance, the gene silencing techniques used for
reducing expression in a plant of an endogenous gene requires the use of
nucleic acid sequences from monocotyledonous plants for transformation of
monocotyledonous plants, and from dicotyledonous plants for
transformation of dicotyledonous plants. Preferably, a nucleic acid
sequence from any given plant species is introduced into that same
species. For example, a nucleic acid sequence from rice is transformed
into a rice plant. However, it is not an absolute requirement that the
nucleic acid sequence to be introduced originates from the same plant
species as the plant in which it will be introduced. It is sufficient
that there is substantial homology between the endogenous target gene and
the nucleic acid to be introduced.

[0139]Described above are examples of various methods for the reduction or
substantial elimination of expression in a plant of an endogenous gene. A
person skilled in the art would readily be able to adapt the
aforementioned methods for silencing so as to achieve reduction of
expression of an endogenous gene in a whole plant or in parts thereof
through the use of an appropriate promoter, for example.

Selectable Marker (Gene)/Reporter Gene

[0140]"Selectable marker", "selectable marker gene" or "reporter gene"
includes any gene that confers a phenotype on a cell in which it is
expressed to facilitate the identification and/or selection of cells that
are transfected or transformed with a nucleic acid construct of the
invention. These marker genes enable the identification of a successful
transfer of the nucleic acid molecules via a series of different
principles. Suitable markers may be selected from markers that confer
antibiotic or herbicide resistance, that introduce a new metabolic trait
or that allow visual selection. Examples of selectable marker genes
include genes conferring resistance to antibiotics (such as nptII that
phosphorylates neomycin and kanamycin, or hpt, phosphorylating
hygromycin, or genes conferring resistance to, for example, bleomycin,
streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,
geneticin (G418), spectinomycin or blasticidin), to herbicides (for
example bar which provides resistance to Basta®; aroA or gox
providing resistance against glyphosate, or the genes conferring
resistance to, for example, imidazolinone, phosphinothricin or
sulfonylurea), or genes that provide a metabolic trait (such as manA that
allows plants to use mannose as sole carbon source or xylose isomerase
for the utilisation of xylose, or antinutritive markers such as the
resistance to 2-deoxyglucose). Expression of visual marker genes results
in the formation of colour (for example β-glucuronidase, GUS or
β-galactosidase with its coloured substrates, for example X-Gal),
luminescence (such as the luciferin/luceferase system) or fluorescence
(Green Fluorescent Protein, GFP, and derivatives thereof). This list
represents only a small number of possible markers. The skilled worker is
familiar with such markers. Different markers are preferred, depending on
the organism and the selection method.

[0141]It is known that upon stable or transient integration of nucleic
acids into plant cells, only a minority of the cells takes up the foreign
DNA and, if desired, integrates it into its genome, depending on the
expression vector used and the transfection technique used. To identify
and select these integrants, a gene coding for a selectable marker (such
as the ones described above) is usually introduced into the host cells
together with the gene of interest. These markers can for example be used
in mutants in which these genes are not functional by, for example,
deletion by conventional methods. Furthermore, nucleic acid molecules
encoding a selectable marker can be introduced into a host cell on the
same vector that comprises the sequence encoding the polypeptides of the
invention or used in the methods of the invention, or else in a separate
vector. Cells which have been stably transfected with the introduced
nucleic acid can be identified for example by selection (for example,
cells which have integrated the selectable marker survive whereas the
other cells die).

[0142]Since the marker genes, particularly genes for resistance to
antibiotics and herbicides, are no longer required or are undesired in
the transgenic host cell once the nucleic acids have been introduced
successfully, the process according to the invention for introducing the
nucleic acids advantageously employs techniques which enable the removal
or excision of these marker genes. One such a method is what is known as
co-transformation. The co-transformation method employs two vectors
simultaneously for the transformation, one vector bearing the nucleic
acid according to the invention and a second bearing the marker gene(s).
A large proportion of transformants receives or, in the case of plants,
comprises (up to 40% or more of the transformants), both vectors. In case
of transformation with Agrobacteria, the transformants usually receive
only a part of the vector, i.e. the sequence flanked by the T-DNA, which
usually represents the expression cassette. The marker genes can
subsequently be removed from the transformed plant by performing crosses.
In another method, marker genes integrated into a transposon are used for
the transformation together with desired nucleic acid (known as the Ac/Ds
technology). The transformants can be crossed with a transposase source
or the transformants are transformed with a nucleic acid construct
conferring expression of a transposase, transiently or stable. In some
cases (approx. 10%), the transposon jumps out of the genome of the host
cell once transformation has taken place successfully and is lost. In a
further number of cases, the transposon jumps to a different location. In
these cases the marker gene must be eliminated by performing crosses. In
microbiology, techniques were developed which make possible, or
facilitate, the detection of such events. A further advantageous method
relies on what is known as recombination systems; whose advantage is that
elimination by crossing can be dispensed with. The best-known system of
this type is what is known as the Cre/lox system. Crel is a recombinase
that removes the sequences located between the loxP sequences. If the
marker gene is integrated between the loxP sequences, it is removed once
transformation has taken place successfully, by expression of the
recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and
REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267;
Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific
integration into the plant genome of the nucleic acid sequences according
to the invention is possible. Naturally, these methods can also be
applied to microorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

[0143]For the purposes of the invention, "transgenic", "transgene" or
"recombinant" means with regard to, for example, a nucleic acid sequence,
an expression cassette, gene construct or a vector comprising the nucleic
acid sequence or an organism transformed with the nucleic acid sequences,
expression cassettes or vectors according to the invention, all those
constructions brought about by recombinant methods in which either
[0144](a) the nucleic acid sequences encoding proteins useful in the
methods of the invention, or [0145](b) genetic control sequence(s) which
is operably linked with the nucleic acid sequence according to the
invention, for example a promoter, or [0146](c) a) and b)are not located
in their natural genetic environment or have been modified by recombinant
methods, it being possible for the modification to take the form of, for
example, a substitution, addition, deletion, inversion or insertion of
one or more nucleotide residues. The natural genetic environment is
understood as meaning the natural genomic or chromosomal locus in the
original plant or the presence in a genomic library. In the case of a
genomic library, the natural genetic environment of the nucleic acid
sequence is preferably retained, at least in part. The environment flanks
the nucleic acid sequence at least on one side and has a sequence length
of at least 50 bp, preferably at least 500 bp, especially preferably at
least 1000 bp, most preferably at least 5000 bp. A naturally occurring
expression cassette--for example the naturally occurring combination of
the natural promoter of the nucleic acid sequences with the corresponding
nucleic acid sequence encoding a polypeptide useful in the methods of the
present invention, as defined above--becomes a transgenic expression
cassette when this expression cassette is modified by non-natural,
synthetic ("artificial") methods such as, for example, mutagenic
treatment. Suitable methods are described, for example, in U.S. Pat. No.
5,565,350 or WO 00/15815.

[0147]A transgenic plant for the purposes of the invention is thus
understood as meaning, as above, that the nucleic acids used in the
method of the invention are not at their natural locus in the genome of
said plant, it being possible for the nucleic acids to be expressed
homologously or heterologously. However, as mentioned, transgenic also
means that, while the nucleic acids according to the invention or used in
the inventive method are at their natural position in the genome of a
plant, the sequence has been modified with regard to the natural
sequence, and/or that the regulatory sequences of the natural sequences
have been modified. Transgenic is preferably understood as meaning the
expression of the nucleic acids according to the invention at an
unnatural locus in the genome, i.e. homologous or, preferably,
heterologous expression of the nucleic acids takes place. Preferred
transgenic plants are mentioned herein.

Transformation

[0148]The term "introduction" or "transformation" as referred to herein
encompasses the transfer of an exogenous polynucleotide into a host cell,
irrespective of the method used for transfer. Plant tissue capable of
subsequent clonal propagation, whether by organogenesis or embryogenesis,
may be transformed with a genetic construct of the present invention and
a whole plant regenerated there from. The particular tissue chosen will
vary depending on the clonal propagation systems available for, and best
suited to, the particular species being transformed. Exemplary tissue
targets include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue (e.g.,
apical meristem, axillary buds, and root meristems), and induced meristem
tissue (e.g., cotyledon meristem and hypocotyl meristem). The
polynucleotide may be transiently or stably introduced into a host cell
and may be maintained non-integrated, for example, as a plasmid.
Alternatively, it may be integrated into the host genome. The resulting
transformed plant cell may then be used to regenerate a transformed plant
in a manner known to persons skilled in the art.

[0149]The transfer of foreign genes into the genome of a plant is called
transformation. Transformation of plant species is now a fairly routine
technique. Advantageously, any of several transformation methods may be
used to introduce the gene of interest into a suitable ancestor cell. The
methods described for the transformation and regeneration of plants from
plant tissues or plant cells may be utilized for transient or for stable
transformation. Transformation methods include the use of liposomes,
electroporation, chemicals that increase free DNA uptake, injection of
the DNA directly into the plant, particle gun bombardment, transformation
using viruses or pollen and microprojection. Methods may be selected from
the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et
al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8:
363-373); electroporation of protoplasts (Shillito R. D. et al. (1985)
Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A
et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle
bombardment (Klein T M et al., (1987) Nature 327: 70) infection with
(non-integrative) viruses and the like. Transgenic plants, including
transgenic crop plants, are preferably produced via
Agrobacterium-mediated transformation. An advantageous transformation
method is the transformation in planta. To this end, it is possible, for
example, to allow the agrobacteria to act on plant seeds or to inoculate
the plant meristem with agrobacteria. It has proved particularly
expedient in accordance with the invention to allow a suspension of
transformed agrobacteria to act on the intact plant or at least on the
flower primordia. The plant is subsequently grown on until the seeds of
the treated plant are obtained (Clough and Bent, Plant J. (1998) 16,
735-743). Methods for Agrobacterium-mediated transformation of rice
include well known methods for rice transformation, such as those
described in any of the following: European patent application EP 1198985
A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant
Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282,
1994), which disclosures are incorporated by reference herein as if fully
set forth. In the case of corn transformation, the preferred method is as
described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996)
or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures
are incorporated by reference herein as if fully set forth. Said methods
are further described by way of example in B. Jenes et al., Techniques
for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and
Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and
in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991)
205-225). The nucleic acids or the construct to be expressed is
preferably cloned into a vector, which is suitable for transforming
Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids
Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then
be used in known manner for the transformation of plants, such as plants
used as a model, like Arabidopsis (Arabidopsis thaliana is within the
scope of the present invention not considered as a crop plant), or crop
plants such as, by way of example, tobacco plants, for example by
immersing bruised leaves or chopped leaves in an agrobacterial solution
and then culturing them in suitable media. The transformation of plants
by means of Agrobacterium tumefaciens is described, for example, by
Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known
inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants;
in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.
Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0150]In addition to the transformation of somatic cells, which then have
to be regenerated into intact plants, it is also possible to transform
the cells of plant meristems and in particular those cells which develop
into gametes. In this case, the transformed gametes follow the natural
plant development, giving rise to transgenic plants. Thus, for example,
seeds of Arabidopsis are treated with agrobacteria and seeds are obtained
from the developing plants of which a certain proportion is transformed
and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet
208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds,
Methods in Arabidopsis Research. Word Scientific, Singapore, pp.
274-289]. Alternative methods are based on the repeated removal of the
inflorescences and incubation of the excision site in the center of the
rosette with transformed agrobacteria, whereby transformed seeds can
likewise be obtained at a later point in time (Chang (1994). Plant J. 5:
551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an
especially effective method is the vacuum infiltration method with its
modifications such as the "floral dip" method. In the case of vacuum
infiltration of Arabidopsis, intact plants under reduced pressure are
treated with an agrobacterial suspension [Bechthold, N (1993). C R Aced
Sci Paris Life Sci, 316: 1194-1199], while in the case of the "floral
dip" method the developing floral tissue is incubated briefly with a
surfactant-treated agrobacterial suspension [Clough, S J and Bent A F
(1998) The Plant J. 16, 735-743]. A certain proportion of transgenic
seeds are harvested in both cases, and these seeds can be distinguished
from non-transgenic seeds by growing under the above-described selective
conditions. In addition the stable transformation of plastids is of
advantages because plastids are inherited maternally is most crops
reducing or eliminating the risk of transgene flow through pollen. The
transformation of the chloroplast genome is generally achieved by a
process which has been schematically displayed in Klaus et al., 2004
[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be
transformed are cloned together with a selectable marker gene between
flanking sequences homologous to the chloroplast genome. These homologous
flanking sequences direct site specific integration into the plastome.
Plastidal transformation has been described for many different plant
species and an overview is given in Bock (2001) Transgenic plastids in
basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312
(3):425-38 or Maliga, P (2003) Progress towards commercialization of
plastid transformation technology. Trends Biotechnol. 21, 20-28. Further
biotechnological progress has recently been reported in form of marker
free plastid transformants, which can be produced by a transient
co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2),
225-229).

T-DNA Activation Tagging

[0151]T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),
involves insertion of T-DNA, usually containing a promoter (may also be a
translation enhancer or an intron), in the genomic region of the gene of
interest or 10 kb up- or downstream of the coding region of a gene in a
configuration such that the promoter directs expression of the targeted
gene. Typically, regulation of expression of the targeted gene by its
natural promoter is disrupted and the gene falls under the control of the
newly introduced promoter. The promoter is typically embedded in a T-DNA.
This T-DNA is randomly inserted into the plant genome, for example,
through Agrobacterium infection and leads to modified expression of genes
near the inserted T-DNA. The resulting transgenic plants show dominant
phenotypes due to modified expression of genes close to the introduced
promoter.

[0153]Homologous recombination allows introduction in a genome of a
selected nucleic acid at a defined selected position. Homologous
recombination is a standard technology used routinely in biological
sciences for lower organisms such as yeast or the moss Physcomitrella.
Methods for performing homologous recombination in plants have been
described not only for model plants (Offring a et al. (1990) EMBO J
9(10): 3077-84) but also for crop plants, for example rice (Terada et al.
(2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin
Biotech 15(2): 132-8).

Yield

[0154]The term "yield" in general means a measurable produce of economic
value, typically related to a specified crop, to an area, and to a period
of time. Individual plant parts directly contribute to yield based on
their number, size and/or weight, or the actual yield is the yield per
square meter for a crop and year, which is determined by dividing total
production (includes both harvested and appraised production) by planted
square meters. The term "yield" of a plant may relate to vegetative
biomass (root and/or shoot biomass), to reproductive organs, and/or to
propagules (such as seeds) of that plant.

Early Vigour

[0155]"Early vigour" refers to active healthy well-balanced growth
especially during early stages of plant growth, and may result from
increased plant fitness due to, for example, the plants being better
adapted to their environment (i.e. optimizing the use of energy resources
and partitioning between shoot and root). Plants having early vigour also
show increased seedling survival and a better establishment of the crop,
which often results in highly uniform fields (with the crop growing in
uniform manner, i.e. with the majority of plants reaching the various
stages of development at substantially the same time), and often better
and higher yield. Therefore, early vigour may be determined by measuring
various factors, such as thousand kernel weight, percentage germination,
percentage emergence, seedling growth, seedling height, root length, root
and shoot biomass and many more.

Increase/Improve/Enhance

[0156]The terms "increase", "improve" or "enhance" are interchangeable and
shall mean in the sense of the application at least a 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, preferably at least 15% or 20%, more preferably 25%, 30%,
35% or 40% more yield and/or growth in comparison to control plants as
defined herein.

Seed Yield

[0157]Increased seed yield may manifest itself as one or more of the
following: a) an increase in seed biomass (total seed weight) which may
be on an individual seed basis and/or per plant and/or per square meter;
b) increased number of flowers per plant; c) increased number of (filled)
seeds; d) increased seed filling rate (which is expressed as the ratio
between the number of filled seeds divided by the total number of seeds);
e) increased harvest index, which is expressed as a ratio of the yield of
harvestable parts, such as seeds, divided by the total biomass; and f)
increased thousand kernel weight (TKW), which is extrapolated from the
number of filled seeds counted and their total weight. An increased TKW
may result from an increased seed size and/or seed weight, and may also
result from an increase in embryo and/or endosperm size.

[0158]An increase in seed yield may also be manifested as an increase in
seed size and/or seed volume. Furthermore, an increase in seed yield may
also manifest itself as an increase in seed area and/or seed length
and/or seed width and/or seed perimeter. Increased yield may also result
in modified architecture, or may occur because of modified architecture.

Greenness Index

[0159]The "greenness index" as used herein is calculated from digital
images of plants. For each pixel belonging to the plant object on the
image, the ratio of the green value versus the red value (in the RGB
model for encoding color) is calculated. The greenness index is expressed
as the percentage of pixels for which the green-to-red ratio exceeds a
given threshold. Under normal growth conditions, under salt stress growth
conditions, and under reduced nutrient availability growth conditions,
the greenness index of plants is measured in the last imaging before
flowering. In contrast, under drought stress growth conditions, the
greenness index of plants is measured in the first imaging after drought.

Plant

[0160]The term "plant" as used herein encompasses whole plants, ancestors
and progeny of the plants and plant parts, including seeds, shoots,
stems, leaves, roots (including tubers), flowers, and tissues and organs,
wherein each of the aforementioned comprise the gene/nucleic acid of
interest. The term "plant" also encompasses plant cells, suspension
cultures, callus tissue, embryos, meristematic regions, gametophytes,
sporophytes, pollen and microspores, again wherein each of the
aforementioned comprises the gene/nucleic acid of interest.

[0162]Surprisingly, it has now been found that modulating expression in a
plant of a nucleic acid encoding the AZ polypeptide from Arabidopsis
(AtAZ) or a homologue thereof gives plants having increased yield
relative to control plants. According to one embodiment of the present
invention, there is provided a method for increasing plant yield,
comprising modulating expression in a plant of a nucleic acid encoding
the AZ polypeptide or a homologue thereof. Advantageously, performance of
the methods according to the present invention results in plants having
increased yield, particularly seed yield, relative to corresponding wild
type plants.

[0163]A "reference", "reference plant", "control", "control plant", "wild
type" or "wild type plant" is in particular a cell, a tissue, an organ, a
plant, or a part thereof, which was not produced according to the method
of the invention. Accordingly, the terms "wild type", "control" or
"reference" are exchangeable and can be a cell or a part of the plant
such as an organelle or tissue, or a plant, which was not modified or
treated according to the herein described method according to the
invention. Accordingly, the cell or a part of the plant such as an
organelle or a plant used as wild type, control or reference corresponds
to the cell, plant or part thereof as much as possible and is in any
other property but in the result of the process of the invention as
identical to the subject matter of the invention as possible. Thus, the
wild type, control or reference is treated identically or as identical as
possible, saying that only conditions or properties might be different
which do not influence the quality of the tested property. That means in
other words that the wild type denotes (1) a plant, which carries the
unaltered or not modulated form of a gene or allele or (2) the starting
material/plant from which the plants produced by the process or method of
the invention are derived.

[0164]Preferably, any comparison between the wild type plants and the
plants produced by the method of the invention is carried out under
analogous conditions. The term "analogous conditions" means that all
conditions such as, for example, culture or growing conditions, assay
conditions (such as buffer composition, temperature, substrates, pathogen
strain, concentrations and the like) are kept identical between the
experiments to be compared.

[0165]The "reference", "control", or "wild type" is preferably a subject,
e.g. an organelle, a cell, a tissue, a plant, which was not modulated,
modified or treated according to the herein described process of the
invention and is in any other property as similar to the subject matter
of the invention as possible. The reference, control or wild type is in
its genome, transcriptome, proteome or metabolome as similar as possible
to the subject of the present invention. Preferably, the term
"reference-" "control-" or "wild type-"-organelle, -cell, -tissue or
plant, relates to an organelle, cell, tissue or plant, which is nearly
genetically identical to the organelle, cell, tissue or plant, of the
present invention or a part thereof preferably 95%, more preferred are
98%, even more preferred are 99,00%, in particular 99,10%, 99,30%,
99,50%, 99,70%, 99,90%, 99,99%, 99, 999% or more. Most preferably the
"reference", "control", or "wild type" is a subject, e.g. an organelle, a
cell, a tissue, a plant, which is genetically identical to the plant,
cell organelle used according to the method of the invention except that
nucleic acid molecules or the gene product encoded by them are changed,
modulated or modified according to the inventive method.

[0166]The term "expression" or "gene expression" is as defined herein,
preferably it results in the appearance of a phenotypic trait as a
consequence of the transcription of a specific gene or specific genes.

[0167]The increase referring to the activity of the polypeptide amounts in
a cell, a tissue, a organelle, an organ or an organism or a part thereof
preferably to at least 5%, preferably to at least 10% or at to least 15%,
especially preferably to at least 20%, 25%, 30% or more, very especially
preferably are to at least 40%, 50% or 60%, most preferably are to at
least 70% or more in comparison to the control, reference or wild type.

[0168]The term "increased yield" as defined herein is taken to mean an
increase in biomass (weight) of one or more parts of a plant, which may
include aboveground (harvestable) parts and/or (harvestable) parts below
ground. In a preferred embodiment, the increased yield is increased seed
yield.

[0169]Therefore, such harvestable parts are preferably seeds, and
performance of the methods of the invention results in plants having
increased seed yield relative to the seed yield of control plants.

[0170]An increase in seed yield may also be manifested as an increase in
seed size and/or seed volume, which may also influence the composition of
seeds (including oil, protein and carbohydrate total content and
composition).

[0171]Taking corn as an example, a yield increase may be manifested as one
or more of the following: increase in the number of plants per square
meter, an increase in the number of ears per plant, an increase in the
number of rows, number of kernels per row, kernel weight, thousand kernel
weight, ear length/diameter, increase in seed filling rate (which is the
number of filled seeds divided by the total number of seeds and
multiplied by 100), among others. Taking rice as an example, a yield
increase may be manifested by an increase in one or more of the
following: number of plants per square meter, number of panicles per
plant, number of spikelets per panicle, number of flowers (florets) per
panicle (which is expressed as a ratio of the number of filled seeds over
the number of primary panicles), increase in the seed filling rate,
(which is the number of filled seeds divided by the total number of seeds
and multiplied by 100), increase in thousand kernel weight, among others.
An increase in yield may also result in modified architecture, or may
occur as a result of modified architecture.

[0172]According to a preferred feature, performance of the methods of the
invention result in plants having increased yield, particularly seed
yield. Therefore, according to the present invention, there is provided a
method for increasing plant yield, which method comprises modulating
expression in a plant of a nucleic acid encoding the AZ polypeptide or a
homologue thereof.

[0173]Since the transgenic plants according to the present invention have
increased yield, it is likely that these plants exhibit an increased
growth rate (during at least part of their life cycle), relative to the
growth rate of control plants at a corresponding stage in their life
cycle. The increased growth rate may be specific to one or more parts of
a plant (including seeds), or may be throughout substantially the whole
plant. Plants having an increased growth rate may have a shorter life
cycle. The life cycle of a plant may be taken to mean the time needed to
grow from a dry mature seed up to the stage where the plant has produced
dry mature seeds, similar to the starting material. This life cycle may
be influenced by factors such as early vigour, growth rate, greenness
index, flowering time and speed of seed maturation. The increase in
growth rate may take place at one or more stages in the life cycle of a
plant or during substantially the whole plant life cycle. Increased
growth rate during the early stages in the life cycle of a plant may
reflect enhanced vigour. The increase in growth rate may alter the
harvest cycle of a plant allowing plants to be sown later and/or
harvested sooner than would otherwise be possible (a similar effect may
be obtained with earlier flowering time). If the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
the same plant species (for example sowing and harvesting of rice plants
followed by sowing and harvesting of further rice plants all within one
conventional growing period). Similarly, if the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
different plants species (for example the planting and harvesting of corn
plants followed by, for example, the planting and optional harvesting of
soy bean, potato or any other suitable plant). Harvesting additional
times from the same rootstock in the case of some crop plants may also be
possible. Altering the harvest cycle of a plant may lead to an increase
in annual biomass production per square meter (due to an increase in the
number of times (say in a year) that any particular plant may be grown
and harvested). An increase in growth rate may also allow for the
cultivation of transgenic plants in a wider geographical area than their
wild-type counterparts, since the territorial limitations for growing a
crop are often determined by adverse environmental conditions either at
the time of planting (early season) or at the time of harvesting (late
season). Such adverse conditions may be avoided if the harvest cycle is
shortened. The growth rate may be determined by deriving various
parameters from growth curves, such parameters may be: T-Mid (the time
taken for plants to reach 50% of their maximal size) and T-90 (time taken
for plants to reach 90% of their maximal size), amongst others.

[0174]According to a preferred feature of the present invention,
performance of the methods of the invention gives plants having an
increased growth rate or increased yield in comparison to control plants.
Therefore, according to the present invention, there is provided a method
for increasing yield and/or growth rate in plants, which method comprises
modulating expression in a plant of a nucleic acid encoding the AZ
polypeptide or a homologue thereof.

[0175]An increase in yield and/or growth rate occurs whether the plant is
under non-stress conditions or whether the plant is exposed to various
stresses compared to control plants. Plants typically respond to exposure
to stress by growing more slowly. In conditions of severe stress, the
plant may even stop growing altogether. Mild stress on the other hand is
defined herein as being any stress to which a plant is exposed which does
not result in the plant ceasing to grow altogether without the capacity
to resume growth. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%, 35% or
30%, preferably less than 25%, 20% or 15%, more preferably less than 14%,
13%, 12%, 11% or 10% or less in comparison to the control plant under
non-stress conditions. Due to advances in agricultural practices
(irrigation, fertilization, pesticide treatments) severe stresses are not
often encountered in cultivated crop plants. As a consequence, the
compromised growth induced by mild stress is often an undesirable feature
for agriculture. Mild stresses are the everyday biotic and/or abiotic
(environmental) stresses to which a plant is exposed. Abiotic stresses
may be due to drought or excess water, anaerobic stress, salt stress,
chemical toxicity, oxidative stress and hot, cold or freezing
temperatures. The abiotic stress may be an osmotic stress caused by a
water stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses caused
by pathogens, such as bacteria, viruses, fungi and insects.

[0176]In particular, the methods of the present invention may be performed
under non-stress conditions or under conditions of mild drought to give
plants having increased yield relative to control plants. As reported in
Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series
of morphological, physiological, biochemical and molecular changes that
adversely affect plant growth and productivity. Drought, salinity,
extreme temperatures and oxidative stress are known to be interconnected
and may induce growth and cellular damage through similar mechanisms.
Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a
particularly high degree of "cross talk" between drought stress and
high-salinity stress. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption of
homeostasis and ion distribution in the cell. Oxidative stress, which
frequently accompanies high or low temperature, salinity or drought
stress, may cause denaturing of functional and structural proteins. As a
consequence, these diverse environmental stresses often activate similar
cell signaling pathways and cellular responses, such as the production of
stress proteins, up-regulation of anti-oxidants, accumulation of
compatible solutes and growth arrest. The term "non-stress" conditions as
used herein are those environmental conditions that do not impose stress,
such as the stresses described above, on plants. Non-stress conditions
allow optimal growth of plants. Persons skilled in the art are aware of
normal soil conditions and climatic conditions for a given location.

[0177]Performance of the methods of the invention gives plants grown under
non-stress conditions or under mild drought conditions increased yield
relative to control plants grown under comparable conditions. Therefore,
according to the present invention, there is provided a method for
increasing yield in plants grown under non-stress conditions or under
mild drought conditions, which method comprises increasing expression in
a plant of a nucleic acid encoding a AZ polypeptide.

[0178]In a preferred embodiment of the invention, the increase in yield
and/or growth rate occurs according to the methods of the present
invention under non-stress conditions.

[0179]Performance of the methods of the invention gives plants grown under
conditions of nutrient deficiency, particularly under conditions of
nitrogen deficiency, increased yield relative to control plants grown
under comparable conditions. Therefore, according to the present
invention, there is provided a method for increasing yield in plants
grown under conditions of nutrient deficiency, which method comprises
increasing expression in a plant of a nucleic acid encoding a AZ
polypeptide. Nutrient deficiency may result from a lack of nutrients such
as nitrogen, phosphates and other phosphorous-containing compounds,
potassium, calcium, cadmium, magnesium, manganese, iron and boron,
amongst others.

[0180]The methods of the invention are advantageously applicable to any
plant. The abovementioned growth characteristics may advantageously be
modified in any plant. Plants that are particularly useful in the methods
of the invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0182]The term "AtAZ polypeptide or a homologue thereof" as defined herein
refers to proteins that comprise at least one ankyrin repeat and at least
one Zinc-finger C3H1 domain, which ankyrin repeat is located upstream of
the C3H1 domain. Preferably, the AZ protein comprises two ankyrin repeats
and two Zinc-finger C3H1 domains such as in the protein represented in
SEQ ID NO: 2. Also preferably, the two ankyrin repeats are located close
to each other. Further preferably, the Zinc-finger C3H1 domains are also
located close to each other and C-terminally of the ankyrin repeats. In
SEQ ID NO: 2, the ankyrin repeats are located on positions D90 to R120
and D125 to L157, the two Zn-finger domains are located on positions H301
to V327 and Q336 to P359 (FIG. 1).

[0183]Also preferably, the AtAZ protein comprises at least one of the
following consensus sequences:

[0185]More preferably, the AtAZ protein comprises two of the
above-mentioned motifs, especially preferably 3 of the above-mentioned
motifs, most preferably all four motifs.

[0186]The ankyrin repeat (SMART SM00248, Interpro IPR002110), as described
in the Interpro database, is one of the most common protein-protein
interaction motifs in nature. Ankyrin repeats are (usually tandemly)
repeated modules of usually about 33 amino acids. They occur in a large
number of functionally diverse proteins mainly from eukaryotes. The few
known examples from prokaryotes and viruses may be the result of
horizontal gene transfers. The repeat has been found in proteins of
diverse function such as transcriptional initiators, cell-cycle
regulators, cytoskeletal, ion transporters and signal transducers. The
ankyrin fold appears to be defined by its structure rather than its
function since there is no specific sequence or structure which is
universally recognised by it. The conserved fold of the ankyrin repeat
unit is known from several crystal and solution structures. Each repeat
folds into a helix-loop-helix structure with a beta-hairpin/loop region
projecting out from the helices at a 90° angle. The repeats stack
together to form an L-shaped structure.

[0187]The Zinc-finger domain ZnF_C3H1 (also known as Znf_CCCH, SMART
SM00356; Interpro IPR000571), as described in the Interpro database is
thought to be involved in DNA-binding. Zinc fingers exist as different
types, depending on the positions of the cysteine residues. Proteins
containing zinc finger domains of the C-x8-C-x5-C-x3-H type (wherein x
represents any amino acid and the digits 8, 5 and 3 represent the number
of amino acids between the conserved C or H residues) include zinc finger
proteins from eukaryotes involved in cell cycle or growth phase-related
regulation, e.g. human TIS11B (butyrate response factor 1), a probable
regulatory protein involved in regulating the response to growth factors,
and the mouse TTP growth factor-inducible nuclear protein, which has the
same function. The mouse TTP protein is induced by growth factors.
Another protein containing this domain is the human splicing factor U2AF
35 kD subunit, which plays a critical role in both constitutive and
enhancer-dependent splicing by mediating essential protein-protein
interactions and protein-RNA interactions required for 3' splice site
selection. It has been shown that different CCCH zinc finger proteins
interact with the 3' untranslated region of various mRNA. This type of
zinc finger is very often present in two copies.

[0188]FIG. 2 describes the consensus sequences as defined in the SMART
database for the ankyrin domain and the zinc-finger domain; however it
should be noted that these consensus sequences might be biased towards
animal protein sequences.

[0190]By aligning other protein sequences with SEQ ID NO: 2, the
corresponding consensus sequences, the C3H1 domain, the ankyrin domain or
other sequence motifs may easily be identified. In this way, AZ
polypeptides or homologues thereof (encompassing orthologues and
paralogues) may readily be identified, using routine techniques well
known in the art, such as by sequence alignment. Methods for the
alignment of sequences for comparison are well known in the art, such
methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the
algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find
the alignment of two complete sequences that maximizes the number of
matches and minimizes the number of gaps. The BLAST algorithm (Altschul
et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence
identity and performs a statistical analysis of the similarity between
the two sequences. The software for performing BLAST analysis is publicly
available through the National Centre for Biotechnology Information.
Homologues may readily be identified using, for example, the ClustalW
multiple sequence alignment algorithm (version 1.83), with the default
pairwise alignment parameters, and a scoring method in percentage. Global
percentages of similarity and identity may also be determined using one
of the methods available in the MatGAT software package (Campanella et
al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that
generates similarity/identity matrices using protein or DNA sequences.).
Minor manual editing may be performed to optimise alignment between
conserved motifs, as would be apparent to a person skilled in the art.
Furthermore, instead of using full-length sequences for the
identification of homologues, specific domains (such as the C3H1 or
ankyrin domain, or one of the motifs defined above) may be used as well.
The sequence identity values, which are indicated below as a percentage
were determined over the entire conserved domain or nucleic acid or amino
acid sequence using the programs mentioned above using the default
parameters.

[0191]Examples of AZ proteins or homologues thereof include the protein
sequences listed in Table A of Example 1.

[0192]It is to be understood that sequences falling under the definition
of "AZ polypeptide or homologue thereof" are not to be limited to the
polypeptide sequences listed in Table A of Example 1, but that any
polypeptide comprising at least one ankyrin repeat, and at least one C3H1
zinc finger domain and preferably also at least one of the consensus
sequence of SEQ ID NO: 3, 4, 5 or 6 as defined above, may be suitable for
use in the methods of the invention. Preferably, the polypeptide is a
polypeptide from Arabidopsis thaliana.

[0193]Encompassed by the term "homologues" are orthologous sequences and
paralogous sequences. Orthologues and paralogues may be found by
performing a so-called reciprocal blast search. This may be done by a
first BLAST involving BLASTing a query sequence (for example, SEQ ID NO:
1 or SEQ ID NO: 2) against any sequence database, such as the publicly
available NCBI database. BLASTN or TBLASTX (using standard default
values) may be used when starting from a nucleotide sequence and BLASTP
or TBLASTN (using standard default values) may be used when starting from
a protein sequence. The BLAST results may optionally be filtered. The
full-length sequences of either the filtered results or non-filtered
results are then BLASTed back (second BLAST) against sequences from the
organism from which the query sequence is derived (where the query
sequence is SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST would
therefore be against Arabidopsis sequences). The results of the first and
second BLASTs are then compared. A paralogue is identified if a
high-ranking hit from the second BLAST is from the same species as from
which the query sequence is derived; an orthologue is identified if a
high-ranking hit is not from the same species as from which the query
sequence is derived. Preferred orthologues are orthologues of SEQ ID NO:
1 or SEQ ID NO: 2. High-ranking hits are those having a low E-value. The
lower the E-value, the more significant the score (or in other words the
lower the chance that the hit was found by chance). Computation of the
E-value is well known in the art. In addition to E-values, comparisons
are also scored by percentage identity. Percentage identity refers to the
number of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length.
Preferably the score is greater than 50, more preferably greater than
100; and preferably the E-value is less than e-5, more preferably less
than e-6. In the case of large families, ClustalW may be used, followed
by the generation of a neighbour joining tree, to help visualize
clustering of related genes and to identify orthologues and paralogues.
Examples of sequences orthologous to SEQ ID NO: 2 include SEQ ID NO: 11,
SEQ ID NO: 15 and SEQ ID NO: 17. SEQ ID NO: 19 is an example of a
paralogue of SEQ ID NO: 2.

[0194]Preferably, the AZ proteins useful in the methods of the present
invention have, besides at least one ankyrin repeat and at least one C3H1
domain, in increasing order of preference, at least 26%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% sequence identity to the protein of SEQ ID NO: 2. The matrix shown in
FIG. 3 (Matrix A) shows similarities and identities (in bold) over the
full-length of various AZ proteins. In case only specific domains are
compared, the identity or similarity may be higher among the different
proteins (Matrix B: comparison of a Zn-finger domain sequence).

[0195]An assay may be carried out to determine AZ activity. DNA-binding
activity and protein-protein interactions may readily be determined in
vitro or in vivo using techniques well known in the art. Examples of in
vitro assays for DNA binding activity include: gel retardation analysis
or yeast one-hybrid assays. An example of an in vitro assay for
protein-protein interactions is the yeast two-hybrid analysis (Fields and
Song (1989) Nature 340:245-6).

[0196]Furthermore, expression of the AZ protein or of a homologue thereof
in plants, and in particular in rice, has the effect of increasing yield
of the transgenic plant when compared to corresponding wild type plants,
wherein increased yield comprises at least one of: total weight of seeds,
total number of seeds and number of filled seeds.

[0197]An AZ polypeptide or homologue thereof is encoded by an AZ nucleic
acid/gene. Therefore the term "AZ nucleic acid/gene" as defined herein is
any nucleic acid/gene encoding an AZ polypeptide or a homologue thereof
as defined above. Examples of AZ nucleic acids include but are not
limited to those represented in Table A of Example 1. AZ nucleic
acids/genes and variants thereof may be suitable in practising the
methods of the invention. Preferably, the variants of an AZ gene
originate from Arabidopsis thaliana. Variant AZ nucleic acid/genes
include portions of an AZ nucleic acid/gene, splice variants, allelic
variants and/or nucleic acids capable of hybridising with an AZ nucleic
acid/gene.

[0198]Reference herein to a "nucleic acid sequence" is taken to mean a
polymeric form of a deoxyribonucleotide or a ribonucleotide polymer of
any length, either double- or single-stranded, or analogues thereof, that
has the essential characteristic of a natural ribonucleotide in that it
can hybridise to nucleic acid sequences in a manner similar to naturally
occurring polynucleotides.

[0199]The term portion as defined herein refers to a piece of DNA encoding
a polypeptide comprising at least one ankyrin repeat and at least one
C3H1 Zn-finger domain. A portion may be prepared, for example, by making
one or more deletions to an AZ nucleic acid. The portions may be used in
isolated form or they may be fused to other coding (or non-coding)
sequences in order to, for example, produce a protein that combines
several activities. When fused to other coding sequences, the resulting
polypeptide produced upon translation may be bigger than that predicted
for the AZ fragment. The portion is typically at least 500, 700 or 900
nucleotides in length, preferably at least 1100, 1300 or 1500 nucleotides
in length, more preferably at least 1700, 1900 or 2100 nucleotides in
length and most preferably at least 2300 or 2400 nucleotides in length.
Preferably, the portion is a portion of a nucleic acid as represented in
Table A of Example 1. Most preferably the portion of an AZ nucleic acid
is as represented by SEQ ID NO: 1 or SEQ ID NO: 53.

[0200]The terms "fragment", "fragment of a sequence" or "part of a
sequence" "portion" or "portion thereof" mean a truncated sequence of the
original sequence referred to. The truncated sequence (nucleic acid or
protein sequence) can vary widely in length; the minimum size being a
sequence of sufficient size to provide a sequence with at least a
comparable function and/or activity of the original sequence referred to
or hybridising with the nucleic acid molecule of the invention or used in
the process of the invention under stringent conditions, while the
maximum size is not critical. In some applications, the maximum size
usually is not substantially greater than that required to provide the
desired activity and/or function(s) of the original sequence. A
comparable function means at least 40%, 45% or 50%, preferably at least
60%, 70%, 80% or 90% or more of the function of the original sequence.

[0201]Another variant of an AZ nucleic acid/gene is a nucleic acid capable
of hybridising under reduced stringency conditions, preferably under
stringent conditions, with an AZ nucleic acid/gene as hereinbefore
defined or with a portion as hereinbefore defined. The hybridizing
sequence is typically at least 300 nucleotides in length, preferably at
least 400 nucleotides in length, more preferably at least 500 nucleotides
in length and most preferably at least 600 nucleotides in length.

[0203]Also useful in the methods of the invention are nucleic acids
encoding homologues (including orthologues or paralogues) of the amino
acid sequence represented by SEQ ID NO: 2, or derivatives thereof.

[0204]Another nucleic acid variant useful in the methods of the present
invention is a splice variant encoding an AZ polypeptide as defined
above. Preferred splice variants are splice variants of the nucleic acid
encoding a polypeptide comprising at least on ankyrin repeat and at least
one C3H1 domain. Preferably, the AZ polypeptide or the homologue thereof
encoded by the splice variant has at least 26%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
sequence identity to SEQ ID NO: 2. Further preferred are splice variants
represented by the nucleic acids represented in Table A of Example 1. For
example, SEQ ID NO: 25 and SEQ ID NO: 47 are encoded by splice variants
of the same gene. Most preferred is the splice variant represented by SEQ
ID NO: 1 or SEQ ID NO: 53.

[0205]Another nucleic acid variant useful in the methods of the present
invention is an allelic variant of a nucleic acid encoding an AZ
polypeptide as defined above. Preferably, the polypeptide encoded by the
allelic variant is represented by the polypeptide sequences listed in
Table A of Example 1. Most preferably, the allelic variant encoding the
AZ polypeptide is represented by SEQ ID NO: 1.

[0206]A further nucleic acid variant useful in the methods of the
invention is a nucleic acid variant obtained by gene shuffling.
Furthermore, site-directed mutagenesis may be used to generate variants
of AZ nucleic acids. Several methods are available to achieve
site-directed mutagenesis; the most common being PCR based methods
(Current Protocols in Molecular Biology. Wiley Eds.).

[0207]Therefore, the invention provides a method for increasing yield
and/or growth rate of a plant, comprising modulating expression in a
plant of a variant of an AZ nucleic acid selected from: [0208](i) a
portion of an AZ nucleic acid; [0209](ii) a nucleic acid hybridising to
an AZ nucleic acid; [0210](iii) a splice variant of a nucleic acid
encoding an AZ polypeptide; [0211](iv) an allelic variant of a nucleic
acid encoding an AZ polypeptide; and [0212](v) a nucleic acid variant
encoding an AZ polypeptide obtained by gene shuffling or site directed
mutagenesis.

[0213]The AZ nucleic acid or variant thereof may be derived from any
natural or artificial source. This nucleic acid may be modified from its
native form in composition and/or genomic environment through deliberate
human manipulation. The nucleic acid is preferably of plant origin,
further preferably from a dicotyledonous species, further preferably from
the family Brassicaceae, more preferably from Arabidopsis thaliana. Most
preferably, the AZ nucleic acid is the Arabidopsis thaliana sequence
represented by SEQ ID NO: 1, and the AZ amino acid sequence is as
represented by SEQ ID NO: 2. Alternatively, the AZ nucleic acid
represented by SEQ ID NO: 53 or the AZ amino acid sequence as represented
by SEQ ID NO: 47 may also be useful in the methods of the present
invention.

[0214]Any reference herein to an AZ polypeptide is therefore taken to mean
an AZ protein as defined above. Any nucleic acid encoding such an AZ
protein is suitable for use in the methods of the invention.

[0215]According to a preferred aspect of the present invention, modulated,
preferably increased expression of the AZ nucleic acid or variant thereof
is envisaged. Methods for increasing the expression of genes or gene
products are well documented in the art. The expression of a nucleic acid
sequence encoding an AZ polypeptide may be modulated by introducing a
genetic modification, which may be introduced, for example, by any one
(or more) of the following methods: T-DNA activation, TILLING,
site-directed mutagenesis, directed evolution and homologous
recombination or by introducing and expressing in a plant a nucleic acid
encoding an AZ polypeptide or a homologue thereof. Following introduction
of the genetic modification, there follows a step of selecting for
modified expression of a nucleic acid encoding an AZ polypeptide or a
homologue thereof, which modification in expression gives plants having
increased yield. Also methods for decreasing the expression of genes or
gene products are known in the art.

[0216]T-DNA activation is described above. A genetic modification may also
be introduced in the locus of an AZ gene using the technique of TILLING
(Targeted Induced Local Lesions In Genomes). The locus of a gene as
defined herein is taken to mean a genomic region, which includes the gene
of interest and 10 kb up- or down stream of the coding region.
Site-directed mutagenesis may be used to generate variants of AZ nucleic
acids. Several methods are available to achieve site-directed
mutagenesis; the most common being PCR based methods (Current Protocols
in Molecular Biology. Wiley Eds.). The effects of the invention may also
be produced using homologous recombination.

[0217]A preferred method for introducing a genetic modification is to
introduce and express in a plant a nucleic acid encoding an AZ
polypeptide or a homologue thereof, as defined above. The nucleic acid to
be introduced into a plant may be a full-length nucleic acid or may be a
portion or a hybridising sequence as hereinbefore defined.

[0218]The invention furthermore provides genetic constructs and vectors to
facilitate introduction and/or expression of the nucleotide sequences
useful in the methods according to the invention.

[0219]Therefore, there is provided a gene construct comprising:
[0220](i) an AZ nucleic acid or variant thereof, as defined hereinabove;
[0221](ii) one or more control sequences operably linked the nucleic acid
sequence of (i);

[0222]Constructs useful in the methods according to the present invention
may be created using recombinant DNA technology well known to persons
skilled in the art. The gene constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for expression of the gene of interest in the
transformed cells. The invention therefore provides use of a gene
construct as defined hereinabove in the methods of the invention.

[0223]Plants are transformed with a vector comprising the sequence of
interest (i.e., a nucleic acid encoding an AZ polypeptide or homologue
thereof). The skilled artisan is well aware of the genetic elements that
must be present on the vector in order to successfully transform, select
and propagate host cells containing the sequence of interest. The
sequence of interest is operably linked to one or more control sequences
(at least to a promoter). The terms "regulatory element", "control
sequence" and "promoter" are defined above. Advantageously, any type of
promoter may be used to drive expression of the nucleic acid sequence.

[0224]Suitable promoters, which are functional in plants, are generally
known. They may take the form of constitutive or inducible promoters.
Suitable promoters can enable the developmental- and/or tissue-specific
expression in multi-cellular eukaryotes; thus, leaf-, root-, flower-,
seed-, stomata-, tuber- or fruit-specific promoters may advantageously be
used in plants.

[0225]Different plant promoters usable in plants are promoters such as,
for example, the USP, the LegB4-, the DC3 promoter or the ubiquitin
promoter from parsley.

[0226]A "plant" promoter comprises regulatory elements, which mediate the
expression of a coding sequence segment in plant cells. Accordingly, a
plant promoter need not be of plant origin, but may originate from
viruses or micro-organisms, in particular for example from viruses which
attack plant cells.

[0227]The "plant" promoter can also originate from a plant cell, e.g. from
the plant which is transformed with the nucleic acid sequence to be
expressed in the inventive process and described herein. This also
applies to other "plant" regulatory signals, for example in "plant"
terminators.

[0229]The expression of plant genes can also be facilitated via a
chemically inducible promoter. Chemically inducible promoters are
particularly suitable when it is desired to express the gene in a
time-specific manner. Examples of such promoters are a salicylic acid
inducible promoter (WO 95/19443), and abscisic acid-inducible promoter
(EP 335 528), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant
J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter (WO
93/21334) or others as described herein.

[0233]According to one preferred feature of the invention, the AZ nucleic
acid or variant thereof is operably linked to a seed-specific promoter.
The seed-specific promoter may be active during seed development and/or
during germination. Seed-specific promoters are well known in the art.
Preferably, the seed-specific promoter is an embryo specific/endosperm
specific/aleurone specific promoter. Further preferably, the
seed-specific promoter drives expression in at least one of: the embryo,
the endosperm, the aleurone. More preferably, the promoter is a WSI18 or
a functionally equivalent promoter. Most preferably, the promoter
sequence is as represented by SEQ ID NO: 9 or SEQ ID NO: 55. It should be
clear that the applicability of the present invention is not restricted
to the AZ nucleic acid represented by SEQ ID NO: 1 or SEQ ID NO: 53, nor
is the applicability of the invention restricted to expression of an AZ
nucleic acid when driven by a seed-specific promoter. Examples of other
seed-specific promoters (embryo specific/endosperm specific/aleurone
specific promoters) which may also be used to drive expression of an AZ
nucleic acid are provided above.

[0234]According to another preferred feature of the invention, the AZ
nucleic acid or variant thereof is operably linked to a constitutive
promoter. A preferred constitutive promoter is a constitutive promoter
that is also substantially ubiquitously expressed. Further preferably the
promoter is derived from a plant, more preferably a monocotyledonous
plant. An example of such a promoter is the GOS2 promoter from rice (SEQ
ID NO: 54 or SEQ ID NO: 56). It should be clear that the applicability of
the present invention is not restricted to the AZ nucleic acid
represented by SEQ ID NO: 1, nor is the applicability of the invention
restricted to expression of a AZ nucleic acid when driven by a
constitutive promoter, and in particular by a GOS2 promoter. Examples of
other constitutive promoters which may also be used to drive expression
of an AZ nucleic acid are shown above.

[0235]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0236]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-ori and colE1.

[0237]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). Therefore, the genetic construct
may optionally comprise a selectable marker gene. Selectable markers are
described in more detail in the "definitions" section herein. The marker
genes may be removed or excised from the transgenic cell once they are no
longer needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section.

[0238]The present invention also encompasses plants, plant parts or plant
cells obtainable by the methods according to the present invention. The
present invention therefore provides plants obtainable by the method
according to the present invention, which plants have introduced therein
an AZ nucleic acid or variant thereof, as defined above.

[0239]The invention also provides a method for the production of
transgenic plants having increased yield, comprising introduction and
expression in a plant of an AZ nucleic acid or a variant thereof as
defined above.

[0240]Host plants for the nucleic acids or the vector used in the method
according to the invention, the expression cassette or construct or
vector are, in principle, advantageously all plants, which are capable of
synthesizing the polypeptides used in the inventive method.

[0241]More specifically, the present invention provides a method for the
production of transgenic plants having increased yield, which method
comprises: [0242](i) introducing and expressing in a plant or plant
cell an AZ nucleic acid or variant thereof; and [0243](ii) cultivating
the plant cell under conditions promoting plant growth and development.

[0244]The nucleic acid may be introduced directly into a plant cell or
into the plant itself (including introduction into a tissue, organ or any
other part of a plant). According to a preferred feature of the present
invention, the nucleic acid is preferably introduced into a plant by
transformation. The terms "introduction" or "transformation" are
described in more detail in the definitions section.

[0245]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant.

[0246]As mentioned, Agrobacteria transformed with an expression vector
according to the invention may also be used in the manner known per se
for the transformation of plants such as plants used as a model, like
Arabidopsis (Arabidopsis thaliana is within the scope of the present
invention not considered as a crop plant), or crop plants, such as
cereals, maize, oats, rye, barley, wheat, soybean, rice, cotton, sugar
beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot,
bell peppers, oilseed rape, tapioca, cassaya, arrow root, tagetes,
alfalfa, lettuce and the various tree, nut, and grapevine species, in
particular oil-containing crop plants such as soy, peanut, castor-oil
plant, sunflower, maize, cotton, flax, oilseed rape, coconut, oil palm,
safflower (Carthamus tinctorius) or cocoa beans, for example by bathing
scarred leaves or leaf segments in an agrobacterial solution and
subsequently culturing them on suitable media.

[0247]The genetically modified plant cells can be regenerated via all
methods with which the skilled worker is familiar. Suitable methods can
be found in the abovementioned publications by S.D. Kung and R. Wu,
Potrykus or Hofgen and Willmitzer.

[0248]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant. To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0249]Following DNA transfer and regeneration, putatively transformed
plants may be evaluated, for instance using Southern analysis, for the
presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels of the
newly introduced DNA may be monitored using Northern and/or Western
analysis, both techniques being well known to persons having ordinary
skill in the art.

[0250]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
and homozygous second-generation (or T2) transformants selected, and the
T2 plants may then further be propagated through classical breeding
techniques.

[0251]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0252]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention. The invention
also includes host cells containing an isolated AZ nucleic acid or
variant thereof. Preferred host cells according to the invention are
plant cells. The invention also extends to harvestable parts of a plant
such as, but not limited to seeds, leaves, fruits, flowers, stem, roots,
rhizomes, tubers and bulbs. The invention furthermore relates to products
directly derived from a harvestable part of such a plant, such as dry
pellets or powders, oil, fat and fatty acids, starch or proteins.

[0253]The present invention also encompasses use of AZ nucleic acids or
variants thereof and use of AZ polypeptides or homologues thereof.

[0255]Nucleic acids encoding AZ polypeptides may find use in breeding
programmes in which a DNA marker is identified which may be genetically
linked to an AZ gene. The nucleic acids/genes may be used to define a
molecular marker. This DNA or protein marker may then be used in breeding
programmes to select plants having increased yield.

[0256]Allelic variants of an AZ nucleic acid/gene may also find use in
marker-assisted breeding programmes. Such breeding programmes sometimes
require introduction of allelic variation by mutagenic treatment of the
plants, using for example EMS mutagenesis; alternatively, the programme
may start with a collection of allelic variants of so called "natural"
origin caused unintentionally. Identification of allelic variants then
takes place, for example, by PCR. This is followed by a step for
selection of superior allelic variants of the sequence in question and
which give increased yield. Selection is typically carried out by
monitoring growth performance of plants containing different allelic
variants of the sequence in question, for example, different allelic
variants of any one of the nucleic acids listed in Table A of Example 1.
Growth performance may be monitored in a greenhouse or in the field.
Further optional steps include crossing plants, in which the superior
allelic variant was identified, with another plant. This could be used,
for example, to make a combination of interesting phenotypic features.

[0257]An AZ nucleic acid may also be used as probes for genetically and
physically mapping the genes that they are a part of, and as markers for
traits linked to those genes. Such information may be useful in plant
breeding in order to develop lines with desired phenotypes. Such use of
AZ nucleic acids or variants thereof requires only a nucleic acid
sequence of at least 15 nucleotides in length. The AZ nucleic acids or
variants thereof may be used as restriction fragment length polymorphism
(RFLP) markers. Southern blots (Sambrook J, Fritsch EF and Maniatis T
(1989) Molecular Cloning, A Laboratory Manual) of restriction-digested
plant genomic DNA may be probed with the AZ nucleic acids. The resulting
banding patterns may then be subjected to genetic analyses using computer
programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in
order to construct a genetic map. In addition, the nucleic acids may be
used to probe Southern blots containing restriction endonuclease-treated
genomic DNAs of a set of individuals representing parent and progeny of a
defined genetic cross. Segregation of the DNA polymorphisms is noted and
used to calculate the position of the AZ nucleic acid in the genetic map
previously obtained using this population (Botstein et al. (1980) Am. J.
Hum. Genet. 32: 314-331).

[0258]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0259]The nucleic acid probes may also be used for physical mapping (i.e.,
placement of sequences on physical maps; see Hoheisel et al. In:
Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,
pp. 319-346, and references cited therein). In another embodiment, the
nucleic acid probes may be used in direct fluorescence in situ
hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
Although current methods of FISH mapping favour use of large clones
(several kb to several hundred kb; see Laan et al. (1995) Genome Res.
5:13-20), improvements in sensitivity may allow performance of FISH
mapping using shorter probes.

[0260]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acids. Examples
include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med
11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et
al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and
Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the
sequence of a nucleic acid is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0261]The methods according to the present invention result in plants
having increased yield, as described hereinbefore. These advantageous
growth characteristics may also be combined with other economically
advantageous traits, such as further yield-enhancing traits, tolerance to
various stresses, traits modifying various architectural features and/or
biochemical and/or physiological features.

Detailed Description for the SYT Polypeptide

[0262]Surprisingly, it has now been found that modulating expression of a
nucleic acid sequence encoding a SYT polypeptide increases plant yield
and/or early vigour under abiotic stress relative to control plants.
Therefore, according to the present invention, there is provided a method
for increasing plant yield and/or early vigour under abiotic stress
relative to control plants, comprising modulating expression in a plant
of a nucleic acid sequence encoding a SYT polypeptide.

[0263]Reference herein to "control plants" is taken to mean any suitable
control plant or plants.

[0264]The "reference", "control", or "wild type" are used herein
interchangeably and is preferably a subject, e.g. an organelle, a cell, a
tissue, a plant, which is as similar to the subject matter of the
invention as possible. The reference, control or wild type is in its
genome, transcriptome, proteome or metabolome as similar as possible to
the subject of the present invention. Preferably, the term "reference-"
"control-" or "wild type-"-organelle, -cell, -tissue or plant, relates to
an organelle, cell, tissue or plant, which is nearly genetically
identical to the organelle, cell, tissue or plant, of the present
invention or to a part thereof, having in increasing order of preference
95%, 98%, 99.00%, 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99,
999% or more genetic identity to the organelle, cell, tissue or plant, of
the present invention. Most preferable the "reference", "control", or
"wild type" is preferably a subject, e.g. an organelle, a cell, a tissue,
a plant, which is genetically identical to the plant, tissue, cell,
organelle used according to the method of the invention except that the
nucleic acid sequences or the gene product in question is changed,
modulated or modified according to the inventive method.

[0265]Preferably, any comparison between the control plants and the plants
produced by the method of the invention is carried out under analogous
conditions. The term "analogous conditions" means that all conditions
such as culture or growing conditions, assay conditions (such as buffer
composition, temperature, substrates, pathogen strain, concentrations and
the like) are kept identical between the experiments to be compared.

[0266]Any reference hereinafter to a "protein useful in the methods of the
invention" is taken to mean a SYT polypeptide as defined herein. Any
reference hereinafter to a "nucleic acid useful in the methods of the
invention" is taken to mean a nucleic acid capable of encoding such a SYT
polypeptide. The nucleic acid to be introduced into a plant (and
therefore useful in performing the methods of the invention) is any
nucleic acid encoding the type of protein which will now be described,
hereafter also named "SYT nucleic acid" or "SYT gene".

[0267]The term "sequence" relates to polynucleotides, nucleic acids,
nucleic acid molecules, peptides, polypeptides and proteins, depending on
the context in which the term "sequence" is used. A "coding sequence" is
a nucleic acid sequence, which is transcribed into mRNA and/or translated
into a polypeptide when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a translation start codon at the 5'-terminus and a
translation stop codon at the 3'-terminus. A coding sequence can include,
but is not limited to mRNA, cDNA, recombinant nucleic acid sequences or
genomic DNA, while introns may be present as well under certain
circumstances. The term "expression" or "gene expression" is as defined
above. The term "modulation" is defined above and means in relation to
expression or gene expression, an increase in expression.

[0272]SYT polypeptides may readily be identified using routine techniques
well known in the art, such as by sequence alignment. Methods for the
alignment of sequences for comparison are well known in the art, such
methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the
algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find
the alignment of two complete sequences that maximizes the number of
matches and minimizes the number of gaps. The BLAST algorithm (Basic
Local Alignment Search Tool; Altschul et al. (1990) J Mol Biol 215:
403-10) calculates percent sequence identity (%) and performs a
statistical analysis of the similarity between the two sequences
(E-value). Percentage identity refers to the number of identical
nucleotides (or amino acids) between the two compared nucleic acid (or
polypeptide) sequences over a particular length. The higher the
similarity between two sequences, the lower the E-value (or in other
words the lower the chance that the hit was found by chance). Computation
of the E-value is well known in the art. The software for performing
BLAST analysis is publicly available through the National Centre for
Biotechnology Information. SYT polypeptides comprising an SNH domain
having in increasing order of preference at least 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ
ID NO: 58 may be identified this way. Alternatively, SYT polypeptides
useful in the methods of the present invention have, in increasing order
of preference, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the
polypeptide of SEQ ID NO: 60. Minor manual editing may be performed to
optimise alignment between conserved motifs, as would be apparent to a
person skilled in the art. In some instances, default parameters may be
adjusted to modify the stringency of the search. For example using BLAST,
the statistical significance threshold (called "expect" value) for
reporting matches against database sequences may be increased to show
less stringent matches. In this way, short nearly exact matches may be
identified. The presence of SEQ ID NO: 146 and of SEQ ID NO: 147 both
comprised in the SYT polypeptides useful in the methods of the invention
may be identified this way.

[0273]Furthermore, the presence of a Met-rich domain or a QG-rich domain
may also readily be identified. As shown in FIG. 7, the Met-rich domain
and QG-rich domain follows the SNH domain. The QG-rich domain may be
taken to be substantially the C-terminal remainder of the polypeptide
(minus the SHN domain); the Met-rich domain is typically comprised within
the first half of the QG-rich (from the N-term to the C-term). Primary
amino acid composition (in %) to determine if a polypeptide domain is
rich in specific amino acids may be calculated using software programs
from the ExPASy server (Gasteiger E et al. (2003) ExPASy: the proteomics
server for in-depth protein knowledge and analysis. Nucleic Acids Res
31:3784-3788), in particular the ProtParam tool. The composition of the
polypeptide of interest may then be compared to the average amino acid
composition (in %) in the Swiss-Prot Protein Sequence data bank (Table
5). Within this databank, the average Met (M) content is of 2.37%, the
average Gln (Q) content is of 3.93% and the average Gly (G) content is of
6.93% (Table 5). As defined herein, a Met-rich domain or a QG-rich domain
has Met content (in %) or a Gln and Gly content (in %) above the average
amino acid composition (in %) in the Swiss-Prot Protein Sequence data
bank. For example in SEQ ID NO: 60, the Met-rich domain at the N-terminal
preceding the SNH domain (from amino acid positions 1 to 24) has Met
content of 20.8% and a QG-rich domain (from amino acid positions 71 to
200) has a Gln (Q) content of 18.6% and a Gly (G) content of 21.4%.
Preferably, the Met domain as defined herein has a Met content (in %)
that is at least 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5,
3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.0, 5.75, 6.0, 6.25, 6.5, 6.75,
7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10 or
more as the average amino acid composition (in %) of said kind of protein
sequences, which are included in the Swiss-Prot Protein Sequence data
bank. Preferably, the QG-rich domain as defined herein has a Gln (Q)
content and/or a Gly (G) content that is at least 1.25, 1.5, 1.75, 2.0,
2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25,
5.0, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5,
8.75, 9.0, 9.25, 9.5, 9.75, 10 or more as much as the average amino acid
composition (in %) of said kind of protein sequences, which are included
in the Swiss-Prot Protein Sequence data bank.

[0275]Examples of nucleic acids encoding SYT polypeptides are given in
Table 6 above. Such nucleic acids are useful in performing the methods of
the invention. The amino acid sequences given in Table 6 above are
example sequences of orthologues and paralogues of the SYT polypeptide
represented by SEQ ID NO: 58, the terms "orthologues" and "paralogues"
being as defined herein. Further orthologues and paralogues may readily
be identified by performing a so-called reciprocal blast search.
Typically, this involves a first BLAST involving BLASTing a query
sequence (for example using any of the sequences listed in Table 6 above)
against any sequence database, such as the publicly available NCB!
database. BLASTN or TBLASTX (using standard default values) are generally
used when starting from a nucleotide sequence, and BLASTP or TBLASTN
(using standard default values) when starting from a protein sequence.
The BLAST results may optionally be filtered. The full-length sequences
of either the filtered results or non-filtered results are then BLASTed
back (second BLAST) against sequences from the organism from which the
query sequence is derived (where the query sequence is SEQ ID NO: 59 or
SEQ ID NO: 60, the second BLAST would therefore be against Arabidopsis
sequences). The results of the first and second BLASTs are then compared.
A paralogue is identified if a high-ranking hit from the first blast is
from the same species as from which the query sequence is derived, a
BLAST back then ideally results in the query sequence amongst the highest
hits; an orthologue is identified if a high-ranking hit in the first
BLAST is not from the same species as from which the query sequence is
derived, and preferably results upon BLAST back in the query sequence
being among the highest hits.

[0276]High-ranking hits are those having a low E-value. The lower the
E-value, the more significant the score (or in other words the lower the
chance that the hit was found by chance). Computation of the E-value is
well known in the art. In addition to E-values, comparisons are also
scored by percentage identity. Percentage identity refers to the number
of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length. In the
case of large families, ClustalW may be used, followed by a neighbour
joining tree, to help visualize clustering of related genes and to
identify orthologues and paralogues.

[0277]It is to be understood that sequences falling under the definition
of a "SYT polypeptide" are not to be limited to the polypeptides given in
Table 6 (and mentioned in the sequence protocol), but that any
polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain
having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence
identity to the SNH domain of SEQ ID NO: 58; and (ii) a Met-rich domain;
and (iii) a QG-rich domain may be suitable in performing the methods of
the invention. Preferably, the SNH domain comprises the residues shown in
black in FIG. 6. Additionally, the SYT polypeptide may comprise one or
more of the following: (a) SEQ ID NO: 146; (b) SEQ ID NO: 147; and (c) a
Met-rich domain at the N-terminal preceding the SNH domain. Most
preferably, the SYT polypeptide is as represented by SEQ ID NO: 60, SEQ
ID NO: 151 or SEQ ID NO: 153.

[0278]A SYT polypeptide typically interacts with GRF (growth-regulating
factor) polypeptides in yeast two-hybrid systems. Yeast two-hybrid
interaction assays are well known in the art (see Field et al. (1989)
Nature 340(6230): 245-246). For example, the SYT polypeptide as
represented by SEQ ID NO: 4 is capable of interacting with AtGRF5 and
with AtGRF9.

[0279]In a further embodiment the invention provides an isolated nucleic
acid sequence comprising a nucleic acid sequence selected from the group
consisting of: [0280](a) an isolated nucleic acid sequence as depicted
in SEQ ID NO: 150 and SEQ ID NO: 152; [0281](b) an isolated nucleic acid
sequence encoding the polypeptide as depicted in SEQ ID NO: 151 and SEQ
ID NO: 153; [0282](c) an isolated nucleic acid sequence whose sequence
can be deduced from a polypeptide as depicted in SEQ ID NO: 151 and SEQ
ID NO: 153 as a result of the degeneracy of the genetic code; [0283](d)
an isolated nucleic acid sequence which encodes a polypeptide which has
at least 70% identity with the polypeptide encoded by the nucleic acid
sequence of (a) to (c); [0284](e) an isolated nucleic acid sequence
encoding a homologue, derivative or active fragment of the polypeptide as
depicted in SEQ ID NO: 151 and SEQ ID NO: 153, which homologue,
derivative or fragment is of plant origin and comprises advantageously
from N-terminal to C-terminal: [0285](i) an SNH domain having in
increasing order of preference at least 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 58;
[0286](ii) a Met-rich domain; [0287](iii) a QG-rich domain;

[0288](f) an isolated nucleic acid sequence capable of hybridising with a
nucleic acid of (a) to (c) above, or its complement, wherein the
hybridising sequence or the complement thereof encodes the plant protein
of (a) to (e);

whereby modified expression of the nucleic acid sequence increases yield
and/or early vigour in plants under abiotic stress compared to control
plants.

[0289]The nucleic acid sequences encoding SYT polypeptides as given in
Table 6, or orthologues or paralogues of any of the aforementioned SEQ ID
NOs need not be full-length nucleic acid sequences, since performance of
the methods of the invention does not rely on the use of full-length
nucleic acid sequences.

[0290]SYT nucleic acid variants may also be suitable in practising the
methods of the invention. Variant SYT nucleic acid sequences typically
are those having the same function as a naturally occurring SYT nucleic
acid sequence, which can be the same biological function or the function
of increasing yield and/or early vigour when expression of the nucleic
acid sequence is modulated in a plant under abiotic stress relative to
control plants. Such variants include portions of a SYT nucleic acid
sequence, splice variants of a SYT nucleic acid sequence, allelic
variants of a SYT nucleic acid sequence, variants of a SYT nucleic acid
sequence obtained by gene shuffling and/or nucleic acid sequences capable
of hybridising with a SYT nucleic acid sequence as defined below.
Preferably, the nucleic acid variant is a variant of a nucleic acid
sequence is as represented by SEQ ID NO: 59, SEQ ID NO: 150 or SEQ ID NO:
152. The terms hybridising sequence, splice variant, allelic variant and
gene shuffling are as described herein.

[0291]Nucleic acids encoding SYT polypeptides need not be full-length
nucleic acids, since performance of the methods of the invention does not
rely on the use of full-length nucleic acid sequences. According to the
present invention, there is provided a method for enhancing yield and/or
early vigour in plants under abiotic stress, comprising introducing and
expressing in a plant a portion of any one of the nucleic acid sequences
given in Table 6, or a portion of a nucleic acid encoding an orthologue,
paralogue or homologue of any of the amino acid sequences given in Table
6.

[0292]The term portion as used herein refers to a piece of DNA encoding a
polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain
having in increasing order of preference at least 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ
ID NO: 58 and (ii) a Met-rich domain; and (iii) a QG-rich domain. A
portion may be prepared, for example, by making one or more deletions to
a SYT nucleic acid sequence. The portions may be used in isolated form or
they may be fused to other coding (or non coding) sequences in order to,
for example, produce a polypeptide that combines several activities. When
fused to other coding sequences, the resulting polypeptide produced upon
translation may be bigger than that predicted for the SYT fragment.
Preferably, the portion is a portion of a nucleic acid sequence as
represented by any one given in Table 6, or orthologues or paralogues of
any of the aforementioned SEQ ID NOs. Most preferably the portion is a
portion of a nucleic acid sequence is as represented by SEQ ID NO: 59,
SEQ ID NO: 150 or SEQ ID NO: 152.

[0293]Another variant of a SYT nucleic acid sequence is a nucleic acid
sequence capable of hybridising under reduced stringency conditions,
preferably under stringent conditions, with a SYT nucleic acid sequence
as hereinbefore defined, which hybridising sequence encodes a SYT
polypeptide or a portion as defined hereinabove.

[0294]According to the present invention, there is provided a method for
enhancing yield and/or early vigour in plants under abiotic stress,
comprising introducing and expressing in a plant a nucleic acid capable
of hybridizing to any one of the nucleic acids given in Table 6, or
comprising introducing and expressing in a plant a nucleic acid capable
of hybridising to a nucleic acid encoding an orthologue, paralogue or
homologue of any of the nucleic acid sequences given in Table 6.

[0295]Hybridising sequences useful in the methods of the invention encode
a SYT polypeptide comprising from N-terminal to C-terminal: (i) an SNH
domain having in increasing order of preference at least 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of
SEQ ID NO: 58 and (ii) a Met-rich domain; and (iii) a QG-rich domain.
Preferably, the hybridising sequence is one that is capable of
hybridising to a nucleic acid sequence as given in Table 6, or
orthologues or paralogues of any of the aforementioned SEQ ID NOs, or to
a portion of any of the aforementioned sequences as defined hereinabove.
Most preferably the hybridizing sequence is one that is capable of
hybridising to a nucleic acid sequence is as represented by SEQ ID NO:
59, SEQ ID NO: 150, SEQ ID NO: 152, or to portions (or probes) thereof.
Methods for designing probes are well known in the art. Probes are
generally less than 1000 by in length, preferably less than 500 by in
length. Commonly, probe lengths for DNA-DNA hybridisations such as
Southern blotting, vary between 100 and 500 bp, whereas the hybridising
region in probes for DNA-DNA hybridisations such as in PCR amplification
generally are shorter than 50 but longer than 10 nucleotides. The
hybridising sequence is typically at least 100, 125, 150, 175, 200 or 225
nucleotides in length, preferably at least 250, 275, 300, 325, 350, 375,
400, 425, 450 or 475 nucleotides in length, further preferably least 500,
525, 550, 575, 600, 625, 650, 675, 700 or 725 nucleotides in length, or
as long as a full length SYT cDNA.

[0296]Another nucleic acid variant useful in the methods of the invention
is a splice variant encoding a SYT polypeptide as defined hereinabove.
Preferred splice variants are splice variants of the SYT nucleic acid
sequences as given in Table 6, or orthologues or paralogues of any of the
aforementioned SEQ ID NOs. Most preferred is a splice variant of a SYT
nucleic acid sequence as represented by SEQ ID NO: 59, SEQ ID NO: 150 or
SEQ ID NO: 152.

[0297]According to the present invention, there is provided a method for
enhancing yield and/or early vigour in plants under abiotic stress,
comprising introducing and expressing in a plant a splice variant of any
one of the nucleic acid sequences given in Table 6, or a splice variant
of a nucleic acid encoding an orthologue, paralogue or homologue of any
of the amino acid sequences given in Table 6.

[0298]Another nucleic acid variant useful in performing the methods of the
invention is an allelic variant of a nucleic acid encoding a SYT
polypeptide as defined hereinabove. Allelic variants exist in nature, and
encompassed within the methods of the present invention is the use of
these natural alleles. Preferred allelic variants are allelic variants of
the SYT nucleic acid sequences as given in Table 6, or orthologues or
paralogues of any of the aforementioned SEQ ID NOs. Most preferred is a
splice variant of a SYT nucleic acid sequence as represented by SEQ ID
NO: 59.

[0299]According to the present invention, there is provided a method for
enhancing yield and/or early vigour in plants under abiotic stress,
comprising introducing and expressing in a plant an allelic variant of
any one of the nucleic acids given in Table 6, or comprising introducing
and expressing in a plant an allelic variant of a nucleic acid encoding
an orthologue, paralogue or homologue of any of the amino acid sequences
given in Table 6.

[0300]A further nucleic acid variant useful in the methods of the
invention is a nucleic acid variant encoding a SYT polypeptide obtained
by gene shuffling (or directed evolution).

[0301]Another nucleic acid variant useful in the methods of the invention
is a nucleic acid variant encoding a SYT polypeptide obtained by
site-directed mutagenesis. Site-directed mutagenesis may be used to
generate variants of SYT nucleic acid sequences. Several methods are
available to achieve site-directed mutagenesis, the most common being PCR
based methods (Current Protocols in Molecular Biology. Wiley Eds).

[0302]According to the present invention, there is provided a method for
enhancing yield and/or early vigour in plants under abiotic stress,
comprising introducing and expressing in a plant a variant of any one of
the nucleic acid sequences given in Table 6, or comprising introducing
and expressing in a plant a variant of a nucleic acid encoding an
orthologue, paralogue or homologue of any of the amino acid sequences
given in Table 6, which variant nucleic acid is obtained by gene
shuffling or site-directed mutagenesis.

[0310]Also useful in the methods of the invention are nucleic acids
encoding homologues of SYT polypeptides as given in Table 6, or
orthologues or paralogues of any of the aforementioned SEQ ID NOs.

[0311]Also useful in the methods of the invention are nucleic acids
encoding derivatives of any one of the SYT nucleic acid sequences as
given in Table 6, or orthologues or paralogues of any of the
aforementioned SEQ ID NOs. Derivatives of orthologues or paralogues of
any of the aforementioned SEQ ID NOs are further examples that may be
suitable for use in the methods of the invention.

[0312]SYT nucleic acid sequences may be derived from any artificial source
or natural source, such as plants, algae, fungi or animals. The nucleic
acid sequence may be modified from its native form in composition and/or
genomic environment through deliberate human manipulation. Preferably the
nucleic acid sequence encoding a SYT polypeptide is of plant origin. The
nucleic acid sequence may be isolated from a dicotyledonous species,
preferably from the family Brassicaceae, further preferably from
Arabidopsis thaliana or Brassica napus. Alternatively, nucleic acid
sequence may be isolated from the family Fabaceae, preferably from
Glycine max. More preferably, the SYT nucleic acid sequence isolated
from: [0313](a) Arabidopsis thaliana is as represented by SEQ ID NO: 59
and the SYT polypeptide as represented by SEQ ID NO: 60; [0314](b)
Brassica napus is as represented by SEQ ID NO: 150 and the SYT
polypeptide as represented by SEQ ID NO: 151; [0315](c) Glycine max is as
represented by SEQ ID NO: 152 and the SYT polypeptide as represented by
SEQ ID NO: 153.

[0316]The terms "yield", "seed yield" and "early vigour" are defined
above. The terms "increased", "improved", "enhanced", "amplified",
"extended", or "rised" are interchangeable and are defined hereinabove.
Taking corn as an example, a yield increase may be manifested as one or
more of the following: increase in the number of plants per square meter,
an increase in the number of ears per plant, an increase in the number of
rows, number of kernels per row, kernel weight, thousand kernel weight,
ear length/diameter, increase in the seed filling rate (which is the
number of filled seeds divided by the total number of seeds and
multiplied by 100), among others. Taking rice as an example, a yield
increase may be manifested by an increase in one or more of the
following: number of plants per square meter, number of panicles per
plant, number of spikelets per panicle, number of flowers (florets) per
panicle (which is expressed as a ratio of the number of filled seeds over
the number of primary panicles), increase in the seed filling rate (which
is the number of filled seeds divided by the total number of seeds and
multiplied by 100), increase in thousand kernel weight, among others.

[0317]An increase in yield may also result in modified architecture, or
may occur as a result of modified architecture.

[0318]According to a preferred feature, performance of the methods of the
invention result in plants having increased biomass, increased seed yield
and/or early vigour under abiotic stress relative to control plants.
Therefore, according to the present invention, there is provided a method
for increasing biomass, seed yield and/or early vigour in a plant under
abiotic stress relative to control plants, which method comprises
modulating expression in a plant of a nucleic acid sequence encoding a
SYT polypeptide. Preferably, by increased biomass is herein taken to mean
the aboveground part (or leafy biomass) during plant development and at
maturity. Preferably, by increased seed yield is herein taken to mean any
one of the following: total seed yield, number of filled seeds, seed fill
rate, TKW and harvest index.

[0319]Since the transgenic plants according to the present invention have
increased yield under abiotic stress, it is likely that these plants
exhibit an increased growth rate under abiotic stress (during at least
part of their life cycle, for example at seedling stage for early
vigour), relative to the growth rate of control plants at a corresponding
stage in their life cycle. The increased growth rate may be specific to
one or more parts of a plant (including seeds), or may be throughout
substantially the whole plant. The increase in growth rate may take place
at one or more stages in the life cycle of a plant or during
substantially the whole plant life cycle. Increased growth rate during
the early stages in the life cycle of a plant may reflect enhanced
vigour. The increase in growth rate may alter the harvest cycle of a
plant allowing plants to be sown later and/or harvested sooner than would
otherwise be possible. If the growth rate is sufficiently increased, it
may allow for the further sowing of seeds of the same plant species (for
example sowing and harvesting of rice plants followed by sowing and
harvesting of further rice plants all within one conventional growing
period). Similarly, if the growth rate is sufficiently increased, it may
allow for the further sowing of seeds of different plants species (for
example the planting and harvesting of corn plants followed by, for
example, the planting and optional harvesting of soybean, potato or any
other suitable plant). Harvesting additional times from the same
rootstock in the case of some crop plants may also be possible. Altering
the harvest cycle of a plant may lead to an increase in annual biomass
production per square meter (due to an increase in the number of times
(say in a year) that any particular plant may be grown and harvested). An
increase in growth rate may also allow for the cultivation of transgenic
plants in a wider geographical area than their wild-type counterparts,
since the territorial limitations for growing a crop are often determined
by adverse environmental conditions either at the time of planting (early
season) or at the time of harvesting (late season). Such adverse
conditions may be avoided if the harvest cycle is shortened. The growth
rate may be determined by deriving various parameters from growth curves,
such parameters may be: T-Mid (the time taken for plants to reach 50% of
their maximal size) and T-90 (time taken for plants to reach 90% of their
maximal size), amongst others. The growth rate of plants is measured
under abiotic stress such as salt stress; water stress (drought or excess
water); reduced nutrient availability stress; temperature stresses caused
by atypical hot or cold/freezing temperatures; oxidative stress; metal
stress; chemical toxicity stress; or combinations thereof.

[0320]Performance of the methods of the invention gives plants having an
increased growth rate under abiotic stress relative to control plants.
Therefore, according to the present invention, there is provided a method
for increasing growth rate in plants under abiotic stress relative to
control plants, which method comprises modulating expression in a plant
of a nucleic acid sequence encoding a SYT polypeptide.

[0321]Plants typically respond to exposure to stress by growing more
slowly. In conditions of severe stress, the plant may even stop growing
altogether. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%, 35% or
30%, preferably less than 25%, 20% or 15%, more preferably less than 14%,
13%, 12%, 11% or 10% or less in comparison to the control plant under
non-stress conditions. Due to advances in agricultural practices
(irrigation, fertilization, pesticide treatments) severe stresses are not
often encountered in cultivated crop plants. As a consequence, the
compromised growth induced by mild stress is often an undesirable feature
for agriculture. Mild stresses are the typical stresses to which a plant
may be exposed. These stresses may be the everyday biotic and/or abiotic
(environmental) stresses to which a plant is exposed. Biotic stresses are
typically those stresses caused by pathogens, such as bacteria, viruses,
fungi, nematodes and insects. Typical abiotic or environmental stresses
comprise any one or more of: salt stress, water stress (drought or excess
water), reduced nutrient availability stress, temperature stresses caused
by atypical hot or cold/freezing temperatures, oxidative stress, metal
stress or chemical toxicity stress.

[0322]Performance of the methods according to the present invention
results in plants having increased yield and/or early vigour under
abiotic stress relative to control plants. As reported in Wang et al.,
(Planta (2003) 218: 1-14), abiotic stress leads to a series of
morphological, physiological, biochemical and molecular changes that
adversely affect plant growth and productivity. Drought, salinity,
extreme temperatures and oxidative stress are often interconnected and
may induce growth and cellular damage through similar mechanisms. For
example, drought and/or salinisation are manifested primarily as osmotic
stress, resulting in the disruption of homeostasis and ion distribution
in the cell. Oxidative stress, which frequently accompanies high or low
temperature, salinity or drought stress, may cause denaturation of
functional and structural proteins. Reduced nutrient availability, in
particular reduced nitrogen availability, is a major limiting factor for
plant growth, for example through the reduced availability of amino acids
for protein synthesis. As a consequence, these diverse environmental
stresses often activate similar cell signaling pathways and cellular
responses, such as the production of stress proteins, up-regulation of
anti-oxidants, accumulation of compatible solutes and growth arrest.

[0323]Since diverse environmental stresses activate similar pathways, the
exemplification of the present invention with salt stress should not be
seen as a limitation to salt stress, but more as a screen to indicate the
involvement of SYT polypeptides in abiotic stresses in general. A review
in TRENDS in Plant Science (Jian-Kang Zhu, Vol. 6, No. 2, February 2001)
confirms that transgenic plants performing better under salt stress often
also perform better under other stresses including chilling, freezing,
heat and drought. A particularly high degree of "cross talk" is reported
between drought stress and high-salinity stress (Rabbani et al., Plant
Physiology, December 2003, Vol. 133, pp. 1755-1767). Therefore, it would
be apparent that a SYT polypeptide (as defined herein) would, along with
its usefulness in increasing yield and/or early vigour in plants under
salt stress, also find use in increasing yield and/or early vigour of the
plant under various other abiotic stresses.

[0324]The term "abiotic stress" as defined herein is taken to mean any one
or more of: salt stress, water stress (drought or excess water), reduced
nutrient availability stress, temperature stresses caused by atypical hot
or cold/freezing temperatures, oxidative stress, metal stress or chemical
toxicity stress. The term salt stress is not restricted to common salt
(NaCl), but may be any one or more of: NaCl, KCl, LiCl, MgCl2,
CaCl2, amongst others.

[0325]Performance of the methods of the invention gives plants having
increased yield and/or early vigour under abiotic stress relative to
control plants. Therefore, according to the present invention, there is
provided a method for increasing plant yield and/or early vigour under
abiotic stress relative to control plants, which method comprises
modulating expression in a plant of a nucleic acid sequence encoding a
SYT polypeptide. By abiotic stress is taken to mean any one or more of:
salt stress, water stress (drought or excess water), reduced nutrient
availability stress, temperature stresses caused by atypical hot or
cold/freezing temperatures, oxidative stress, metal stress or chemical
toxicity stress.

[0326]The methods of the invention are advantageously applicable to any
plant. Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0327]A preferred method for introducing a genetic modification is to
introduce and express in a plant a nucleic acid sequence encoding a SYT
polypeptide. A SYT polypeptide is defined as a polypeptide comprising
from N-terminal to C-terminal: (i) an SNH domain having in increasing
order of preference at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
sequence identity to the SNH domain of SEQ ID NO: 58; and (ii) a Met-rich
domain; and (iii) a QG-rich domain. Preferably, the SNH domain comprises
the residues shown in black in FIG. 6. Further preferably, the SNH domain
is represented by SEQ ID NO: 57.

[0328]According to a preferred aspect of the present invention, the
modulated expression of a SYT nucleic acid sequence is increased
expression. The increase in expression may lead to raised SYT mRNA or
polypeptide levels, which could equate to raised activity of the SYT
polypeptide; or the activity may also be raised when there is no change
in polypeptide levels, or even when there is a reduction in polypeptide
levels. This may occur when the intrinsic properties of the SYT
polypeptide are altered, for example, by making mutant versions that are
more active that the wild type polypeptide. Methods for increasing or
reducing expression of genes or gene products are known in the art.

[0329]The invention also provides genetic constructs and vectors to
facilitate introduction and/or expression of the nucleotide sequences
useful in the methods according to the invention.

[0330]Therefore, there is provided a genetic construct comprising:
[0331](i) A nucleic acid sequence encoding a SYT polypeptide, as defined
hereinabove, or a nucleic acid sequence as represented by SEQ ID NO: 150
or SEQ ID NO: 152; [0332](ii) One or more control sequences capable of
driving expression of the nucleic acid sequence of (i); and optionally
[0333](iii) A transcription termination sequence.

[0334]A preferred construct is one where the control sequence is a
promoter derived from a plant, preferably from a monocotyledonous plant
if a monocotyledonous is to be transformed.

[0335]Constructs useful in the methods according to the present invention
may be constructed using recombinant DNA technology well known to persons
skilled in the art. The genetic constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for expression of the gene of interest in the
transformed cells. The invention therefore provides use of a genetic
construct as defined hereinabove in the methods of the invention.

[0336]Plants are transformed with a vector comprising the sequence of
interest (i.e., a nucleic acid sequence encoding a SYT polypeptide). The
sequence of interest is operably linked to one or more control sequences
(at least to a promoter).

[0337]Advantageously, any type of promoter may be used to drive expression
of the nucleic acid sequence.

[0343]In one embodiment, the SYT nucleic acid sequence is operably linked
to a constitutive promoter. A constitutive promoter is transcriptionally
active during most, but not necessarily all, phases of its growth and
development and is substantially ubiquitously expressed. Preferably the
promoter is derived from a plant, more preferably the promoter is from a
monocotyledonous plant if a monocotyledonous plant is to be transformed.
Further preferably, the constitutive promoter is a GOS2 promoter that is
represented by a nucleic acid sequence substantially similar to SEQ ID
NO: 145 or SEQ ID NO: 56. Most preferably the GOS2 promoter is as
represented by SEQ ID NO: 56 or SEQ ID NO: 145. It should be clear that
the applicability of the present invention is not restricted to the SYT
nucleic acid sequence represented by SEQ ID NO: 59, nor is the
applicability of the invention restricted to expression of a SYT nucleic
acid sequence when driven by a GOS2 promoter. Examples of other
constitutive promoters that may also be used to drive expression of a SYT
nucleic acid sequence are shown in the definitions section.

[0344]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0345]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-ori and colE1.

[0346]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). Therefore, the genetic construct
may optionally comprise a selectable marker gene. Selectable markers are
described in more detail in the "definitions" section herein. The marker
genes may be removed or excised from the transgenic cell once they are no
longer needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section.

[0347]The present invention also encompasses plants obtainable by the
methods according to the present invention. The present invention
therefore provides plants, plant parts and plant cells obtainable by the
methods according to the present invention, which plants have introduced
therein a SYT nucleic acid sequence and which plants, plant parts and
plant cells are preferably from a crop plant, further preferably from a
monocotyledonous plant.

[0348]The invention also provides a method for the production of
transgenic plants having increased yield and/or early vigour under
abiotic stress, comprising introduction and expression in a plant of a
SYT nucleic acid sequence.

[0349]More specifically, the present invention provides a method for the
production of transgenic plants, preferably monocotyledonous plants,
having increased yield and/or early vigour under abiotic stress, which
method comprises: [0350](i) introducing and expressing in a plant or
plant cell a nucleic acid sequence encoding a SYT polypeptide; and
[0351](ii) cultivating the plant cell under conditions promoting plant
growth and development.

[0352]The nucleic acid of (i) may be any of the nucleic acids capable of
encoding a SYT polypeptide, as defined hereinabove, or a nucleic acid
sequence as represented by SEQ ID NO: 150 or SEQ ID NO: 152.

[0353]Subsequent generations of the plants obtained from cultivating step
(ii) may be propagated by a variety of means, such as by clonal
propagation or classical breeding techniques. For example, a first
generation (or T1) transformed plant may be selfed to give homozygous
second generation (or T2) transformants, and the T2 plants further
propagated through classical breeding techniques.

[0354]The nucleic acid sequence may be introduced directly into a plant
cell or into the plant itself (including introduction into a tissue,
organ or any other part of a plant). According to a preferred feature of
the present invention, the nucleic acid sequence is introduced into a
plant by transformation.

[0355]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant. To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0356]Following DNA transfer and regeneration, putatively transformed
plants may also be evaluated, for instance using Southern analysis, for
the presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels of the
newly introduced DNA may be monitored using Northern and/or Western
analysis, both techniques being well known to persons having ordinary
skill in the art.

[0357]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
to give homozygous second generation (or T2) transformants, and the T2
plants further propagated through classical breeding techniques.

[0358]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0359]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention. The invention
also includes host cells containing an isolated SYT nucleic acid
sequence. Preferred host cells according to the invention are plant
cells. The invention also extends to harvestable parts of a plant such
as, but not limited to seeds, leaves, fruits, flowers, stem cultures,
roots, rhizomes, tubers and bulbs. The invention furthermore relates to
products derived, preferably directly derived, from a harvestable part of
such a plant, such as dry pellets or powders, meal, oil, fat and fatty
acids, starch or proteins.

[0360]According to a preferred feature of the invention, the modulated
expression is increased expression. Methods for increasing expression of
nucleic acids or genes, or gene products, are well documented in the art
and examples are provided in the definitions section.

[0361]Alternatively, the expression of a nucleic acid sequence encoding a
SYT polypeptide may be modulated by introducing a genetic modification,
for example, by any one (or more) of the following techniques: T-DNA
activation, TILLING, homologous recombination, or by introducing and
expressing in a plant a nucleic acid sequence encoding a SYT polypeptide.
Following introduction of the genetic modification, there follows a step
of selecting for modulated expression of a nucleic acid sequence encoding
a SYT polypeptide, which modulated expression gives plants having
increased yield and/or early vigour under abiotic stress.

[0362]One such technique is T-DNA activation tagging. The promoter to be
introduced may be any promoter capable of driving expression of a gene in
the desired organism, in this case a plant. For example, constitutive,
tissue-preferred, cell type-preferred and inducible promoters are all
suitable for use in T-DNA activation. The effects of the invention may
also be reproduced using the technique of TILLING (Targeted Induced Local
Lesions In Genomes). The effects of the invention may also be reproduced
using homologous recombination.

[0363]The present invention also encompasses use of SYT nucleic acid
sequences and use of SYT polypeptides, and use of a construct as defined
hereinabove in increasing plant yield and/or early vigour under abiotic
stress.

[0364]SYT nucleic acid sequences or SYT polypeptides may find use in
breeding programmes in which a DNA marker is identified that may be
genetically linked to a SYT. The SYT nucleic acid sequences or SYT
polypeptides may be used to define a molecular marker. This DNA or
polypeptide marker may then be used in breeding programmes to select
plants having increased yield. The SYT gene may, for example, be a
nucleic acid sequence as represented by any one of the SYT nucleic acid
sequences as given in Table 6, or orthologues or paralogues of any of the
aforementioned SEQ ID NOs.

[0365]Allelic variants of a SYT nucleic acid sequence may also find use in
marker-assisted breeding programmes. Such breeding programmes sometimes
require introduction of allelic variation by mutagenic treatment of the
plants, using for example EMS mutagenesis; alternatively, the programme
may start with a collection of allelic variants of so called "natural"
origin caused unintentionally. Identification of allelic variants then
takes place, for example, by PCR. This is followed by a step for
selection of superior allelic variants of the sequence in question and
which give increased yield. Selection is typically carried out by
monitoring growth performance of plants containing different allelic
variants of the sequence in question, for example, different allelic
variants of any one of the SYT nucleic acid sequences as given in Table
6, or orthologues or paralogues of any of the aforementioned SEQ ID NOs.
Growth performance may be monitored in a greenhouse or in the field.
Further optional steps include crossing plants, in which the superior
allelic variant was identified, with another plant. This could be used,
for example, to make a combination of interesting phenotypic features.

[0366]SYT nucleic acid sequences may also be used as probes for
genetically and physically mapping the genes that they are a part of, and
as markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. Such use of SYT nucleic acid sequences requires only a
nucleic acid sequence of at least 15 nucleotides in length. The SYT
nucleic acid sequences may be used as restriction fragment length
polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and
Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of
restriction-digested plant genomic DNA may be probed with the SYT nucleic
acid sequences. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander et al.
(1987) Genomics 1: 174-181) in order to construct a genetic map. In
addition, the nucleic acid sequences may be used to probe Southern blots
containing restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic cross.
Segregation of the DNA polymorphisms is noted and used to calculate the
position of the SYT nucleic acid sequence in the genetic map previously
obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet.
32:314-331).

[0367]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0369]In another embodiment, the nucleic acid probes may be used in direct
fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favor use of
large clones (several kb to several hundred kb; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow performance
of FISH mapping using shorter probes.

[0370]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acid sequences.
Examples include allele-specific amplification (Kazazian (1989) J. Lab.
Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS;
Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation
(Landegren et al. (1988) Science 241:1077-1080), nucleotide extension
reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping
(Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods,
the nucleic acid sequence is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0371]The methods according to the present invention result in plants
having increased yield under abiotic stress, as described hereinbefore.
These yield-enhancing traits may also be combined with other economically
advantageous traits, such as further yield-enhancing traits, tolerance to
various stresses, traits modifying various architectural features and/or
biochemical and/or physiological features.

Detailed Description for the cpFBPase Polypeptide

[0372]Surprisingly, it has now been found that increasing expression in
aboveground parts of a plant of a nucleic acid sequence encoding a
chloroplastic fructose-1,6-bisphosphatase (cpFBPase) polypeptide
increases plant yield relative to control plants. Therefore, according to
the present invention, there is provided a method for increasing plant
yield relative to control plants, comprising increasing expression in
aboveground parts of a plant of a nucleic acid sequence encoding a
cpFBPase polypeptide.

[0373]Reference herein to "control plants" is taken to mean any suitable
control plant or plants. The "reference", "control", or "wild type" are
used herein interchangeably and is preferably a subject, e.g. an
organelle, a cell, a tissue, a plant, which is as similar to the subject
matter of the invention as possible. The reference, control or wild type
is in its genome, transcriptome, proteome or metabolome as similar as
possible to the subject of the present invention. Preferably, the term
"reference-" "control-" or "wild type-"-organelle, -cell, -tissue or
plant, relates to an organelle, cell, tissue or plant, which is nearly
genetically identical to the organelle, cell, tissue or plant, of the
present invention or a part thereof, preferably 95%, 98%, 99,00%, 99,10%,
99,30%, 99,50%, 99,70%, 99,90%, 99,99%, 99, 999% or more identical. Most
preferably the "reference", "control", or "wild type" is preferably a
subject, e.g. an organelle, a cell, a tissue, a plant, which is
genetically identical to the plant, tissue, cell, organelle used
according to the method of the invention except that the nucleic acid
sequences or the gene product encoded by them are changed, modulated or
modified according to the inventive method.

[0374]Unless otherwise specified, the terms "polynucleotides", "nucleic
acid" and "nucleic acid molecule", are interchangeably in the present
context. Unless otherwise specified, the terms "peptide", "polypeptide"
and "protein" are interchangeably in the present context. The term
"sequence" may relate to polynucleotides, nucleic acids, nucleic acid
molecules, peptides, polypeptides and proteins, depending on the context
in which the term "sequence" is used. The terms "gene(s)",
"polynucleotide", "nucleic acid sequence", "nucleotide sequence", or
"nucleic acid molecule(s)" as used herein refers to a polymeric form of
nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. The terms refer only to the primary structure of
the molecule.

[0375]Thus, the terms "nucleic acid sequence", "gene(s)",
"polynucleotide", "nucleotide sequence", or "nucleic acid molecule(s)" as
used herein include double- and single-stranded DNA and RNA. They also
include known types of modifications, for example, methylation, "caps",
substitutions of one or more of the naturally occurring nucleotides with
an analog. Preferably, the DNA or RNA sequence of the invention comprises
a coding sequence encoding the herein defined polypeptide.

[0376]A "coding sequence" is a nucleic acid sequence, which is transcribed
into mRNA and/or translated into a polypeptide when placed under the
control of appropriate regulatory sequences. The boundaries of the coding
sequence are determined by a translation start codon at the 5'-terminus
and a translation stop codon at the 3'-terminus. A coding sequence can
include, but is not limited to mRNA, cDNA, recombinant nucleic acid
sequences or genomic DNA, while introns may be present as well under
certain circumstances.

[0377]The term "chloroplastic fructose-1,6-bisphosphatase (cpFBPase)
polypeptide" as defined herein refers to a polypeptide functioning in the
chloroplast and comprising: (i) at least one FBPase domain; and (ii) at
least one redox regulatory insertion.

[0379]It is to be understood that sequences falling under the definition
of a "cpFBPase polypeptide" are not to be limited to the polypeptides
given in Table 7 (and mentioned herein above) but that any polypeptide
functioning in the chloroplast and comprising: (i) at least one FBPase
domain; and (ii) at least one redox regulatory insertion, may be suitable
in performing the methods of the invention. Preferably, the cpFBPase
polypeptide is as represented by SEQ ID NO: 155.

[0380]However, performance of the invention is not restricted to these
sequences; the methods of the invention may advantageously be performed
using any cpFBPase-encoding nucleic acid or cpFBPase polypeptide as
defined herein.

[0381]Examples of nucleic acids encoding cpFBPase polypeptides are given
in Table C of Example 12 herein. Such nucleic acids are useful in
performing the methods of the invention. The amino acid sequences given
in Table C of Example 12 are example sequences of orthologues and
paralogues of the cpFBPase polypeptide represented by SEQ ID NO: 155, the
terms "orthologues" and "paralogues" being as defined herein. Orthologues
and paralogues may easily be found by performing a so-called reciprocal
blast search. This may be done by a first BLAST involving BLASTing a
query sequence (for example, SEQ ID NO: 154 or SEQ ID NO: 155) against
any sequence database, such as the publicly available NCB! database.
BLASTN or TBLASTX (using standard default values) may be used when
starting from a nucleotide sequence and BLASTP or TBLASTN (using standard
default values) may be used when starting from a polypeptide sequence.
The BLAST results may optionally be filtered. The full-length sequences
of either the filtered results or non-filtered results are then BLASTed
back (second BLAST) against sequences from the organism from which the
query sequence is derived (where the query sequence is SEQ ID NO: 154 or
SEQ ID NO: 155, the second BLAST would therefore be against Chlamydomonas
sequences). The results of the first and second BLASTs are then compared.
A paralogue is identified if a high-ranking hit from the first BLAST is
from the same species as from which the query sequence is derived, a
BLAST back then ideally results in the query sequence as highest hit
(besides itself); an orthologue is identified if a high-ranking hit in
the first BLAST is not from the same species as from which the query
sequence is derived and preferably results upon BLAST back in the query
sequence amongst the highest hits. High-ranking hits are those having a
low E-value. The lower the E-value, the more significant the score (or in
other words the lower the chance that the hit was found by chance).
Computation of the E-value is well known in the art. In addition to
E-values, comparisons are also scored by percentage identity. Percentage
identity refers to the number of identical nucleotides (or amino acids)
between the two compared nucleic acid (or polypeptide) sequences over a
particular length. An example detailing the identification of orthologues
and paralogues is given in Example 12. In the case of large families,
ClustalW may be used, followed by a neighbour joining tree, to help
visualize clustering of related genes and to identify orthologues and
paralogues. Preferably, cpFBPase polypeptides useful in the methods of
the invention are functioning in the chloroplast and comprise: (i) at
least one FBPase domain; and (ii) at least one redox regulatory
insertion; and (iii) in increasing order of preference, at least 40%,
45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity
to SEQ ID NO: 155 (calculations shown in Example 14).

[0382]The alignment of multiple polypeptide sequences is used to find
conserved domains and characteristic motifs in protein families, in the
determination of evolutionary linkage and in the improved prediction of
secondary and tertiary structure. Many programs are available to a person
skilled in the art to perform such analysis, for example, the ones
proposed by the Expasy proteomics toolbox hosted by the Swiss Institute
for Bioinformatics.

[0383]Methods for the alignment of sequences for comparison are well known
in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:
443-453) to find the global (i.e. spanning the complete sequences)
alignment of two sequences that maximizes the number of matches and
minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990)
J Mol Biol 215: 403-10) calculates percent sequence identity and performs
a statistical analysis of the similarity between the two sequences. The
software for performing BLAST analysis is publicly available through the
National Centre for Biotechnology Information (NCBI). Homologues may
readily be identified using, for example, the ClustalW multiple sequence
alignment algorithm (version 1.83), with the default pairwise alignment
parameters, and a scoring method in percentage. Global percentages of
similarity and identity may also be determined using one of the methods
available in the MatGAT software package (Campanella et al., BMC
Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates
similarity/identity matrices using protein or DNA sequences.). Minor
manual editing may be performed to optimise alignment between conserved
motifs, as would be apparent to a person skilled in the art. Furthermore,
instead of using full-length sequences for the identification of
homologues, specific domains may also be used. The sequence identity
values may be determined over the entire nucleic acid or amino acid
sequence or over selected domains or conserved motif(s), using the
programs mentioned above using the default parameters.

[0384]The terms "domain" and "motif" are described in the definitions
section. Special databases exisit for the identification of domains. The
FBPase domain in a cpFBPase polypeptide may be identified using, for
example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,
5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; hosted by
the EMBL at Heidelberg, Germany), InterPro (Mulder et al., (2003) Nucl.
Acids. Res. 31, 315-318; hosted by the European Bioinformatics Institute
(EBI) in the United Kingdom), Prosite (Bucher and Bairoch (1994), A
generalized profile syntax for biomolecular sequences motifs and its
function in automatic sequence interpretation. (In) ISMB-94; Proceedings
2nd International Conference on Intelligent Systems for Molecular
Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp
53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:
D134-D137, (2004), The ExPASy proteomics server is provided as a service
to the scientific community (hosted by the Swiss Institute of
Bioinformatics (SIB) in Switzerland) or Pfam (Bateman et al., Nucleic
Acids Research 30(1): 276-280 (2002), hosted by the Sanger Institute in
the United Kingdom). For example, in the InterPro database, the FBPase
domain comprised in the cpFBPase polypeptide is designated IPR000146. In
Example 15 are listed the entries identified in a number of databases,
related to a cpFBPase polypeptide as represented by SEQ ID NO : 155.

[0385]An important motif comprised within the FBPase domain is the redox
regulatory insertion, which is present only the cpFBPase polypeptides and
not in the cyFBPase polypeptides. By aligning all FBPases polypeptides
using the methods described hereinabove and in Example 13 (and FIG. 12),
an insertion of amino acids comprising at least two cysteine residues
necessary for disulphide bridge formation (i.e. redox regulation) is
identified. The conserved cysteines are named after their position in the
mature pea (Pisum sativa) polypeptide, i.e. Cys153, Cys173 and Cys178.
Cys153 and Cys173 usually are the two partners involved in disulphide
bridge formation (Chiadmi et al. (1999) EMBO J 18(23): 6809-6815). Cys153
and Cys173 are separated by a loop whose length is of 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues,
preferably of 14, 15, 16, 17, 18, or 19 amino acid residues.

[0386]The task of protein subcellular localisation prediction is important
and well studied. Knowing a protein's localisation helps elucidate its
function. Experimental methods for protein localization range from
immunolocalization to tagging of proteins using green fluorescent protein
(GFP). Such methods are accurate although labor-intensive compared with
computational methods. Recently much progress has been made in
computational prediction of protein localisation from sequence data.
Among algorithms well known to a person skilled in the art are available
at the ExPASy Proteomics tools hosted by the Swiss Institute for
Bioinformatics, for example, PSort, TargetP, ChloroP, Predotar, LipoP,
MITOPROT, PATS, PTS1, SignalP and others. The identification of
subcellular localisation of the polypeptide of the invention is shown in
Example 16. In particular SEQ ID NO: 155 of the present invention is
assigned to the plastidic (chloroplastic) compartment of photosynthetic
(autotrophic) cells.

[0387]Methods for targeting to plastids are well known in the art and
include the use of transit peptides. Table 7 below shows examples of
transit peptides which can be used to target any FBPase polypeptide to a
plastid, which FBPase polypeptide is not, in its natural form, normally
targeted to a plastid, or which FBPase polypeptide in its natural form is
targeted to a plastid by virtue of a different transit peptide (for
example, its natural transit peptide). For example a nucleic acid
sequence encoding a cyFBPase may also be suitable for use in the methods
of the invention so long as the nucleic acid is targeted to a plastid,
preferably to a chloroplast, and that is comprises at least one
regulatory redox insertion.

[0388]cpFBPase polypeptides as represented by SEQ ID NO: 155 are enzymes
with as Enzyme Commission (EC; classification of enzymes by the reactions
they catalyse) number EC 3.1.3.11 for fructose-bisphosphatase (also
called D-fructose-1,6-bisphosphate 1-phosphohydrolase). cpFBPase
polypeptides catalyze the irreversible conversion of
fructose-1,6-bisphophate to fructose-6-phosphate and Pi. The functional
assay may be an assay for cpFBPase activity based on a colorimetric Pi
assay, as described by Huppe and Buchanan (1989) in Naturforsch. 44c:
487-494. Other methods to assay the enzymatic activity are described by
Alscher-Herman (1982) in Plant Physiol 70: 728-734.

[0389]By "functioning in the chloroplast" is taken to mean herein that the
cpFBPase polypeptide is active in the chloroplast, i.e., the cpFBPase
polypeptide is performing the enzymatic reaction consisting in
hydrolysing fructose-1,6-bisphosphate into fructose-6-phosphate and Pi,
in the chloroplast.

[0390]The nucleic acid sequences encoding cpFBpase polypeptides as given
in Table 7, or encoding orthologues or paralogues of any of the
aforementioned SEQ ID NOs need not be full-length nucleic acid sequences,
since performance of the methods of the invention does not rely on the
use of full-length nucleic acid sequences.

[0391]cpFBPase nucleic acid variants may also be suitable in practising
the methods of the invention. Variant cpFBPase nucleic acid sequences
typically are those having the same function as a naturally occurring
cpFBPase nucleic acid sequence, which can be the same biological function
or the function of increasing yield when expression of the nucleic acid
sequence is increased in aboveground parts of a plant relative to a
control plant. Examples of such cpFBPase variants include portions of
nucleic acid sequences, nucleic acid sequences capable of hybridising to
cpFBPases, splice variants, allelic variants either naturally occurring
or by DNA manipulation, a cpFBPase nucleic acid sequence obtained by gene
shuffling, or a cpFBPase nucleic acid sequence obtained by site-directed
mutagenesis.

[0392]The term "portion" as used herein refers to a piece of DNA encoding
a polypeptide functioning in the chloroplast and comprising: (i) at least
one FBPase domain; and (ii) at least one redox regulatory insertion.

[0393]A portion may be prepared, for example, by making one or more
deletions to a nucleic acid encoding a cpFBPase polypeptide as defined
hereinabove. The portions may be used in isolated form or they may be
fused to other coding (or non coding) sequences in order to, for example,
produce a polypeptide that combines several activities. In another
example, the naturally occurring transit peptide coding sequence may be
replaced by a transit peptide coding sequence from another photosynthetic
organism, or by a synthetic one. If chloroplast transformation is
considered, the transit peptide coding sequence may be removed
altogether. When fused to other coding sequences, the resultant
polypeptide produced upon translation may be bigger than that predicted
for the cpFBPase portion. Portions useful in the methods of the invention
are typically at least 900 nucleotides in length, preferably at least
1000 nucleotides in length, more preferably at least 1100 nucleotides in
length and most preferably at least 1200 nucleotides in length.
Preferably, the portion is a portion of a nucleic acid sequence as
represented by any one of the nucleic acid sequences given in Table 7, or
nucleic acid sequences encoding orthologues or paralogues of any of the
aforementioned SEQ ID NOs. Most preferably the portion is a portion of a
nucleic acid sequence as represented by SEQ ID NO: 154.

[0394]According to the present invention, there is provided a method for
increasing yield in plants, comprising increasing expression in a plant
of a portion of any one of the nucleic acid sequences given in Table C of
Example 12, or of a portion of a nucleic acid encoding an orthologue,
paralogue or homologue of any of the amino acid sequences given in Table
C of Example 12.

[0395]Another nucleic acid variant useful in the methods of the invention,
is a nucleic acid capable of hybridising under reduced stringency
conditions, preferably under stringent conditions, with a nucleic acid
sequence encoding a cpFBPase polypeptide as defined hereinabove, or a
with a portion as defined hereinabove.

[0396]According to the present invention, there is provided a method for
increasing yield in plants, comprising increasing expression in a plant
of a nucleic acid capable of hybridizing to any one of the nucleic acids
given in Table C of Example 12, or comprising increasing expression in a
plant of a nucleic acid capable of hybridising to a nucleic acid encoding
an orthologue, paralogue or homologue of any of the nucleic acid
sequences given in Table C of Example 12.

[0397]Hybridising sequences useful in the methods of the invention, encode
a cpFBPase polypeptide functioning in the chloroplast and comprising: (i)
at least one FBPase domain; and (ii) at least one redox regulatory
insertion, and having substantially the same biological activity as the
cpFBPase polypeptide as represented by SEQ ID NO: 155. Methods for
designing probes are well known in the art. The hybridising sequence is
typically less than 1000 by in length, preferably less than 900, 800,
700, 600 or 500 by in length. Commonly, hybridising sequence lengths for
DNA-DNA hybridisations such as Southern blotting vary between 100 and 500
bp, whereas for DNA-DNA hybridisations such as in PCR amplification
generally shorter than 50 but longer than 10 nucleotides. Preferably, the
hybridising sequence is one that is capable of hybridising to any of the
nucleic acid sequences (or to probes derived from) as represented by the
nucleic acid sequences given in Table 7, or nucleic acid sequences
encoding orthologues or paralogues of any of the aforementioned SEQ ID
NOs, or to a portion of any of the aforementioned sequences, a portion
being as defined above. Most preferably the hybridising sequence is
capable of hybridising to SEQ ID NO: 154, or to portions (or probes)
thereof.

[0398]Another nucleic acid variant useful in the methods of the invention
is a splice variant encoding a cpFBPase polypeptide as defined
hereinabove. Such variants will be ones in which the biological activity
of the protein is substantially retained; this may be achieved by
selectively retaining functional segments of the protein. Preferred
splice variants are splice variants of the cpFBPase nucleic acid
sequences as given in Table 7, or nucleic acid sequences encoding
orthologues or paralogues of any of the aforementioned SEQ ID NOs. Most
preferred is a splice variant of a cpFBPase nucleic acid sequence as
represented by SEQ ID NO: 154.

[0399]Another nucleic acid variant useful in performing the methods of the
invention is an allelic variant of a nucleic acid sequence encoding a
cpFBPase polypeptide as defined hereinabove. Allelic variants exist in
nature, and encompassed within the methods of the present invention is
the use of these natural alleles. Preferred allelic variants are allelic
variants of the cpFBPase nucleic acid sequences as given in Table 7, or
nucleic acid sequences encoding orthologues or paralogues of any of the
aforementioned SEQ ID NOs. Most preferred is a splice variant of a
cpFBPase nucleic acid sequence as represented by SEQ ID NO: 154.

[0400]According to the present invention, there is provided a method for
increasing yield in plants, comprising increasing expression in a plant
of a splice variant of any one of the nucleic acid sequences given in
Table C of Example 12, or of a splice variant of a nucleic acid encoding
an orthologue, paralogue or homologue of any of the amino acid sequences
given in Table C of Example 12.

[0401]A further nucleic acid variant useful in the methods of the
invention is a nucleic acid variant encoding a cpFBPase polypeptide
obtained by gene shuffling (or directed evolution). Most preferred is a
nucleic acid variant obtained by gene shuffling of a cpFBPase nucleic
acid sequence as represented by SEQ ID NO: 155.

[0402]According to the present invention, there is provided a method for
increasing yield in plants, comprising increasing expression in a plant
of a variant of any one of the nucleic acid sequences given in Table C of
Example 12, or comprising increasing expression in a plant of a variant
of a nucleic acid encoding an orthologue, paralogue or homologue of any
of the amino acid sequences given in Table C of Example 12, which variant
nucleic acid is obtained by gene shuffling.

[0403]Another nucleic acid variant useful in the methods of the invention
is a nucleic acid variant encoding a cpFBPase polypeptide obtained by
site-directed mutagenesis. Site-directed mutagenesis may be used to
generate variants of cpFBPase nucleic acid sequences. Several methods are
available to achieve site-directed mutagenesis, the most common being PCR
based methods (Current Protocols in Molecular Biology, Wiley Eds). For
example, a mutation affecting the disulfide bridge formation is to change
one of the conserved cysteines into serine, thereby making cpFBPase
constitutively active (Chiadmi et al. (1999) EMBO J 18(23): 6809-6815).
Most preferred is a nucleic acid variant obtained by site-directed
mutagenesis of a cpFBPase nucleic acid sequence as represented by SEQ ID
NO: 154.

[0411]Also useful in the methods of the invention are nucleic acid
sequences encoding homologues of cpFBPase polypeptides as given in Table
7, or encoding orthologues or paralogues of any of the aforementioned SEQ
ID NOs.

[0412]Also useful in the methods of the invention are nucleic acid
sequences encoding derivatives of any one of the cpFBPase polypeptides as
given in Table 7, or orthologues or paralogues of any of the
aforementioned SEQ ID NOs. Derivatives of cpFBPase polypeptides as
represented by any one given in Table 7, or orthologues or paralogues of
any of the aforementioned SEQ ID NOs are further examples that may be
suitable for use in the methods of the invention.

[0414]Performance of the methods of the invention gives plants having
enhanced yield. The terms "yield", "increased", "improved", "enhanced",
"amplified", "extended", "augmented" or "rised" are interchangeable and
are defined above. Increased biomass may manifest itself as increased
root biomass. Increased root biomass may be due to increased number of
roots, increased root thickness and/or increased root length. Increased
yield may manifest itself as one or more of the following: [0415](i)
increased biomass (weight) of one or more parts of a plant, particularly
aboveground (harvestable) parts, increased root biomass or increased
biomass of any other harvestable part; [0416](ii) increased early vigour,
defined herein as the seedling aboveground area three weeks
post-germination; [0417](iii) increased total seed yield, which includes
an increase in seed biomass (seed weight) and which may be an increase in
the seed weight per plant or on an individual seed basis; [0418](iv)
increased number of panicles per plant; [0419](v) increased number of
flowers ("florets") per panicle; [0420](vi) increased seed fill rate;
[0421](vii) increased number of (filled) seeds; [0422](viii) increased
seed size (length, width area, perimeter), which may also influence the
composition of seeds; [0423](ix) increased seed volume, which may also
influence the composition of seeds; [0424](x) increased harvest index,
which is expressed as a ratio of the yield of harvestable parts, such as
seeds, over the total biomass; and [0425](xi) increased thousand kernel
weight (TKW), which is extrapolated from the number of filled seeds
counted and their total weight. An increased TKW may result from an
increased seed size and/or seed weight. An increased TKW may result from
an increase in embryo size and/or endosperm size.

[0426]An increase in seed size, seed volume, seed area, seed perimeter,
seed width and seed length may be due to an increase in specific parts of
a seed, for example due to an increase in the size of the embryo and/or
endosperm and/or aleurone and/or scutellum, or other parts of a seed.

[0427]In particular, increased yield is increased seed yield, and is
selected from one or more of the following: (i) increased seed weight;
(ii) increased number of filled seeds; (iii) increased seed fill rate;
and (iv) increased harvest index.

[0428]Taking corn as an example, a yield increase may be manifested as one
or more of the following: increase in the number of plants per square
meter, an increase in the number of ears per plant, an increase in the
number of rows, number of kernels per row, kernel weight, thousand kernel
weight, ear length/diameter, increase in the seed filling rate (which is
the number of filled seeds divided by the total number of seeds and
multiplied by 100), among others.

[0429]Taking rice as an example, a yield increase may be manifested by an
increase in one or more of the following: number of plants per square
meter, number of panicles per plant, number of spikelets per panicle,
number of flowers (florets) per panicle (which is expressed as a ratio of
the number of filled seeds over the number of primary panicles), increase
in the seed filling rate (which is the number of filled seeds divided by
the total number of seeds and multiplied by 100), increase in thousand
kernel weight, among others.

[0430]An increase in yield may also result in modified architecture, or
may occur as a result of modified architecture.

[0431]According to a preferred feature, performance of the methods of the
invention results in plants having increased seed yield relative to
control plants. Therefore, according to the present invention, there is
provided a method for increasing seed yield, which method comprises
increasing expression in aboveground parts of a plant of a nucleic acid
sequence encoding a cpFBPase polypeptide.

[0432]Since the transgenic plants according to the present invention have
increased yield, it is likely that these plants exhibit an increased
growth rate (during at least part of their life cycle), relative to the
growth rate of control plants at a corresponding stage in their life
cycle.

[0433]The increased growth rate may be specific to one or more parts of a
plant (including seeds), or may be throughout substantially the whole
plant. Plants having an increased growth rate may have a shorter life
cycle. The life cycle of a plant may be taken to mean the time needed to
grow from a dry mature seed up to the stage where the plant has produced
dry mature seeds, similar to the starting material. This life cycle may
be influenced by factors such as early vigour, growth rate, greenness
index, flowering time and speed of seed maturation. The increase in
growth rate may take place at one or more stages in the life cycle of a
plant or during substantially the whole plant life cycle. Increased
growth rate during the early stages in the life cycle of a plant may
reflect enhanced vigour. The increase in growth rate may alter the
harvest cycle of a plant allowing plants to be sown later and/or
harvested sooner than would otherwise be possible (a similar effect may
be obtained with earlier flowering time). If the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
the same plant species (for example sowing and harvesting of rice plants
followed by sowing and harvesting of further rice plants all within one
conventional growing period). Similarly, if the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
different plants species (for example the sowing and harvesting of corn
plants followed by, for example, the sowing and optional harvesting of
soybean, potato or any other suitable plant). Harvesting additional times
from the same rootstock in the case of some crop plants may also be
possible. Altering the harvest cycle of a plant may lead to an increase
in annual biomass production per square meter (due to an increase in the
number of times (say in a year) that any particular plant may be grown
and harvested). An increase in growth rate may also allow for the
cultivation of transgenic plants in a wider geographical area than their
wild-type counterparts, since the territorial limitations for growing a
crop are often determined by adverse environmental conditions either at
the time of planting (early season) or at the time of harvesting (late
season). Such adverse conditions may be avoided if the harvest cycle is
shortened. The growth rate may be determined by deriving various
parameters from growth curves, such parameters may be: T-Mid (the time
taken for plants to reach 50% of their maximal size) and T-90 (time taken
for plants to reach 90% of their maximal size), amongst others.

[0434]According to a preferred feature of the present invention,
performance of the methods of the invention gives plants having an
increased growth rate relative to control plants. Therefore, according to
the present invention, there is provided a method for increasing the
growth rate of plants, which method comprises modulating expression,
preferably increasing expression, in a plant of a nucleic acid encoding a
cpFBPase polypeptide as defined herein.

[0435]An increase in yield and/or growth rate occurs whether the plant is
under non-stress conditions or whether the plant is exposed to various
stresses compared to control plants. Plants typically respond to exposure
to stress by growing more slowly. In conditions of severe stress, the
plant may even stop growing altogether. Mild stress on the other hand is
defined herein as being any stress to which a plant is exposed which does
not result in the plant ceasing to grow altogether without the capacity
to resume growth. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%, 35% or
30%, preferably less than 25%, 20% or 15%, more preferably less than 14%,
13%, 12%, 11% or 10% or less in comparison to the control plant under
non-stress conditions. Due to advances in agricultural practices
(irrigation, fertilization, pesticide treatments) severe stresses are not
often encountered in cultivated crop plants. As a consequence, the
compromised growth induced by mild stress is often an undesirable feature
for agriculture. Mild stresses are the everyday biotic and/or abiotic
(environmental) stresses to which a plant is exposed. Abiotic stresses
may be due to drought or excess water, anaerobic stress, salt stress,
chemical toxicity, oxidative stress and hot, cold or freezing
temperatures. The abiotic stress may be an osmotic stress caused by a
water stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses caused
by pathogens, such as bacteria, viruses, fungi and insects.

[0436]In particular, the methods of the present invention may be performed
under non-stress conditions or under conditions of mild drought to give
plants having increased yield relative to control plants. As reported in
Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series
of morphological, physiological, biochemical and molecular changes that
adversely affect plant growth and productivity. Drought, salinity,
extreme temperatures and oxidative stress are known to be interconnected
and may induce growth and cellular damage through similar mechanisms.
Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a
particularly high degree of "cross talk" between drought stress and
high-salinity stress. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption of
homeostasis and ion distribution in the cell. Oxidative stress, which
frequently accompanies high or low temperature, salinity or drought
stress, may cause denaturing of functional and structural proteins. As a
consequence, these diverse environmental stresses often activate similar
cell signalling pathways and cellular responses, such as the production
of stress proteins, up-regulation of anti-oxidants, accumulation of
compatible solutes and growth arrest. The term "non-stress" conditions as
used herein are those environmental conditions that allow optimal growth
of plants. Persons skilled in the art are aware of normal soil conditions
and climatic conditions for a given location.

[0437]Performance of the methods of the invention gives plants grown under
non-stress conditions or under mild drought conditions increased yield
relative to control plants grown under comparable conditions. Therefore,
according to the present invention, there is provided a method for
increasing yield in plants grown under non-stress conditions or under
mild drought conditions, which method comprises increasing expression in
a plant of a nucleic acid encoding a cpFBPase polypeptide.

[0438]Performance of the methods of the invention gives plants grown under
conditions of nutrient deficiency, particularly under conditions of
nitrogen deficiency, increased yield relative to control plants grown
under comparable conditions. Therefore, according to the present
invention, there is provided a method for increasing yield in plants
grown under conditions of nutrient deficiency, which method comprises
increasing expression in a plant of a nucleic acid encoding a cpFBPase
polypeptide. Nutrient deficiency may result from a lack of nutrients such
as nitrogen, phosphates and other phosphorous-containing compounds,
potassium, calcium, cadmium, magnesium, manganese, iron and boron,
amongst others.

[0439]Preferably the increase in yield and/or growth rate occurs according
to the method of invention under non-stress or mild abiotic or mild
biotic stress conditions.

[0440]The term "expression" or "gene expression" means the transcription
of a specific gene or specific genes or specific genetic construct. The
term "expression" or "gene expression" in particular means the
transcription of a gene or genes or genetic construct into structural RNA
(rRNA, tRNA) or mRNA with or without subsequent translation of the latter
into a protein. The process includes transcription of DNA and processing
of the resulting mRNA product.

[0442]The term "aboveground parts of plant" is herein taken to mean plant
parts excluding the roots, root hairs and any other plant part that is in
the soil, i.e., that is not directly exposed to light.

[0443]By increasing the expression (in a plastid) of a nucleic acid
sequence encoding a cpFBPase polypeptide, an increase in the amount of
cpFBPase polypeptide is obtained. This increase in amount of cpFBPase
polypeptide (in a plastid) leads to an increase in cpFBPase activity.
Alternatively, activity may also be increased when there is no change in
the amount of a cpFBPase polypeptide, or even when there is a reduction
in the amount of a cpFBPase polypeptide. This may occur when the
intrinsic properties of the polypeptide are altered, for example, by
making mutant versions that are more active than the wild type
polypeptide.

[0444]The expression of a nucleic acid sequence encoding a cpFBPase
polypeptide is increased in a plastid using techniques well known in the
art, such as by targeting a cpFBPase polypeptide to the plastid using
transit peptide sequences or by direct transformation of a cpFBPase
polypeptide without transit peptide sequences, into a plastid. Expression
may be increased in any plastid, however, preferred is preferentially
increasing expression in a chloroplast.

[0445]The expression of a nucleic acid sequence encoding a cpFBPase
polypeptide may be modulated by introducing a genetic modification, for
example, by any one (or more) of the following techniques: T-DNA
activation, TILLING, homologous recombination, or by introducing and
expressing in a plant a nucleic acid sequence encoding a cpFBPase
polypeptide. Following introduction of the genetic modification, there
follows a step of selecting for increased expression of a nucleic acid
sequence encoding a cpFBPase polypeptide, which increased expression
gives plants having increased yield relative to control plants.

[0446]One such technique is T-DNA activation tagging. The promoter to be
introduced may be any promoter capable of driving expression of a gene in
the desired organism, in this case a plant. For example, constitutive,
aboveground parts, below ground parts, tissue-preferred, cell
type-preferred and inducible promoters are all suitable for use in T-DNA
activation. The effects of the invention may also be reproduced using the
technique of TILLING or using homologous recombination.

[0447]A preferred method for introducing a genetic modification is to
introduce and express in aboveground parts of a plant a nucleic acid
sequence encoding a cpFBPase polypeptide. The cpFBPase as defined herein
refers to a polypeptide functioning in the chloroplast and comprising:
(i) at least one FBPase domain; and (ii) at least one redox regulatory
insertion.

[0448]In one embodiment of the present invention, the expression of a
nucleic acid sequence encoding a cpFBPase polypeptide is increased
expression (in aboveground parts of plant). The increase in expression
may lead to raised cpFBPase mRNA or polypeptide levels, which could
equate to raised activity of the cpFBPase polypeptide; or the activity
may also be raised when there is no change in polypeptide levels, or even
when there is a reduction in polypeptide levels. This may occur when the
intrinsic properties of the cpFBPase polypeptide are altered, for
example, by making mutant versions that are more active that the wild
type polypeptide. Methods for increasing expression of genes or gene
products are well documented in the art.

[0449]The invention also provides genetic constructs and vectors to
facilitate introduction and/or expression of the nucleotide sequences
useful in the methods according to the invention.

[0450]Therefore, there is provided a genetic construct comprising:
[0451](i) A nucleic acid sequence encoding a cpFBPase polypeptide, as
defined hereinabove; [0452](ii) One or more control sequences capable of
driving expression in aboveground parts of a plant, of the nucleic acid
sequence of (i); and optionally [0453](iii) A transcription termination
sequence;

[0454]A preferred construct is one whether the control sequence is a
promoter derived from a plant, preferably from a monocotyledonous plant
if a monocotyledonous is to be transformed.

[0455]Constructs useful in the methods according to the present invention
may be constructed using recombinant DNA technology well known to persons
skilled in the art. The genetic constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for expression of the gene of interest in the
transformed cells. The invention therefore provides use of a genetic
construct as defined hereinabove in the methods of the invention.

[0456]Plants are transformed with a vector comprising the sequence of
interest (i.e., a nucleic acid sequence encoding a cpFBPase polypeptide).
The sequence of interest is operably linked to one or more control
sequences (at least to a promoter). The terms "regulatory element",
"control sequence" and "promoter" are all used interchangeably herein and
are defined above.

[0457]Advantageously, the promoter used to drive expression of the nucleic
acid sequence may be a tissue-preferred promoter, i.e. one that is
capable of preferentially initiating transcription in certain tissues,
such as the leaves, stems, seed tissue etc. Promoters able to initiate
transcription in certain tissues only are referred to herein as
"tissue-specific", similarly, promoters able to initiate transcription in
certain cells only are referred to herein as "cell-specific".
Additionally or alternatively, the promoter may occur natural or
synthetic. Preferably, the promoter is capable of driving expression of a
nucleic acid encoding a cpFBPase polypeptide in aboveground parts of a
plant, i.e., in parts exposed to light for proper cpFBPase redox
regulation.

[0462]Other promoters which are available for the expression of genes in
plants are leaf-specific promoters such as those described in DE-A
19644478 or light-regulated promoters such as, for example, the pea petE
promoter.

[0463]In one embodiment, the nucleic acid sequence is operably linked to a
constitutive promoter. A constitutive promoter is transcriptionally
active during most, but not necessarily all, phases of its growth and
development and is substantially ubiquitously expressed (including in
aboveground parts of a plant). Preferably the promoter is derived from a
plant, more preferably the promoter is from a monocotyledonous plant if a
monocotyledonous plant is to be transformed. Further preferably, the
constitutive promoter is a GOS2 promoter that is represented by a nucleic
acid sequence substantially similar to SEQ ID NO: 208 or SEQ ID NO: 56.
Most preferably the GOS2 promoter is as represented by SEQ ID NO: 56 or
SEQ ID NO: 208. It should be clear that the applicability of the present
invention is not restricted to the cpFBPase nucleic acid sequence as
represented by SEQ ID NO: 154, nor is the applicability of the invention
restricted to expression of a cpFBPase nucleic acid sequence when driven
by a GOS2 promoter. Examples of other constitutive promoters that may
also be used to drive expression of a cpFBPase nucleic acid sequence are
shown above.

[0464]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0465]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-ori and colE1.

[0466]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). Therefore, the genetic construct
may optionally comprise a selectable marker gene. Selectable markers are
described in more detail in the "definitions" section herein. The marker
genes may be removed or excised from the transgenic cell once they are no
longer needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section. Different
markers are preferred, depending on the organism and the selection
method.

[0467]The present invention also encompasses plants (including seeds)
obtainable by the methods according to the present invention. The present
invention therefore provides plants, plant parts and plant cells
obtainable by the methods according to the present invention, which
plants have introduced therein a cpFBPase nucleic acid sequence and which
plants, plant parts and plant cells are preferably from a crop plant,
further preferably from a monocotyledonous plant.

[0468]The invention also provides a method for the production of
transgenic plants having increased yield relative to control plants,
comprising introduction and expression in a plant of a nucleic acid
sequence encoding a cpFBPase polypeptide.

[0469]More specifically, the present invention provides a method for the
production of transgenic plants, preferably monocotyledonous plants,
having increased yield relative to control plants, which method
comprises: [0470](i) introducing and expressing in aboveground parts of
a plant or plant cell a nucleic acid sequence encoding a cpFBPase
polypeptide; and [0471](ii) cultivating the plant cell under conditions
promoting plant growth and development.

[0472]The nucleic acid of (i) may be any of the nucleic acids capable of
encoding a cpFBPase polypeptide as defined herein.

[0473]The nucleic acid sequence may be introduced directly into a plant
cell or into the plant itself (including introduction into a tissue,
organ or any other part of a plant). According to a preferred feature of
the present invention, the nucleic acid sequence is introduced into a
plant by transformation.

[0474]The genetically modified plant cells can be regenerated via all
methods with which the skilled worker is familiar. Suitable methods can
be found in the abovementioned publications by S.D. Kung and R. Wu,
Potrykus or Hofgen and Willmitzer.

[0475]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant. To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0476]Following DNA transfer and regeneration, putatively transformed
plants may be evaluated, for instance using Southern analysis, for the
presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels of the
newly introduced DNA may be monitored using Northern and/or Western
analysis, quantitative PCR, such techniques being well known to persons
having ordinary skill in the art.

[0477]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
to give homozygous second generation (or T2) transformants, and the T2
plants further propagated through classical breeding techniques.

[0478]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0479]The methods of the invention are advantageously applicable to any
plant. Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0481]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention. The invention
also includes host cells containing an isolated cpFBPase nucleic acid
sequence. Preferred host cells according to the invention are plant
cells. The invention also extends to harvestable parts of a plant such
as, but not limited to seeds, leaves, fruits, flowers, stem cultures,
rhizomes, tubers and bulbs. The invention furthermore relates to products
derived, preferably directly derived, from a harvestable part of such a
plant, such as dry pellets or powders, meal, oil, fat and fatty acids,
starch or proteins.

[0482]The present invention also encompasses use of cpFBPase nucleic acid
sequences and use of cpFBPase polypeptides, and use of a construct as
defined hereinabove in increasing plant yield relative to control plants.
The increased plant yield is in particular increased seed yield. By
increased seed yield is herein taken to mean any one of the following:
(i) increased seed weight; (ii) increased number of filled seeds; (iii)
increased seed fill rate; and (iv) increased harvest index.

[0483]cpFBPase nucleic acid sequences or cpFBPase polypeptides may find
use in breeding programmes in which a DNA marker is identified that may
be genetically linked to a cpFBPase locus. The cpFBPase nucleic acid
sequences or cpFBPase polypeptides may be used to define a molecular
marker. This DNA or polypeptide marker may then be used in breeding
programmes to select plants having increased yield. The cpFBPase gene
may, for example, be a nucleic acid sequence as represented by any one of
the cpFBPase nucleic acid sequences as given in Table 7, or nucleic acid
sequences encoding orthologues or paralogues of any of the aforementioned
SEQ ID NOs.

[0484]Allelic variants of a cpFBPase nucleic acid sequence may also find
use in marker-assisted breeding programmes. Such breeding programmes
sometimes require introduction of allelic variation by mutagenic
treatment of the plants, using for example EMS mutagenesis;
alternatively, the programme may start with a collection of allelic
variants of so called "natural" origin caused unintentionally.
Identification of allelic variants then takes place, for example, by PCR.
This is followed by a step for selection of superior allelic variants of
the sequence in question and which give increased yield. Selection is
typically carried out by monitoring growth performance of plants
containing different allelic variants of the sequence in question, for
example, different allelic variants of any one of the cpFBPase nucleic
acid sequences as given in Table 7, or nucleic acid sequences encoding
orthologues or paralogues of any of the aforementioned SEQ ID NOs. Growth
performance may be monitored in a greenhouse or in the field. Further
optional steps include crossing plants, in which the superior allelic
variant was identified, with another plant. This could be used, for
example, to make a combination of interesting phenotypic features.

[0485]cpFBPase nucleic acid sequences may also be used as probes for
genetically and physically mapping the genes that they are a part of, and
as markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. Such use of cpFBPase nucleic acid sequences requires only a
nucleic acid sequence of at least 15 nucleotides in length. The cpFBPase
nucleic acid sequences may be used as restriction fragment length
polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and
Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of
restriction-digested plant genomic DNA may be probed with the cpFBPase
nucleic acid sequences. The resulting banding patterns may then be
subjected to genetic analyses using computer programs such as MapMaker
(Lander et al. (1987) Genomics 1: 174-181) in order to construct a
genetic map. In addition, the nucleic acid sequences may be used to probe
Southern blots containing restriction endonuclease-treated genomic DNAs
of a set of individuals representing parent and progeny of a defined
genetic cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the cpFBPase nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et al.
(1980) Am. J. Hum. Genet. 32:314-331).

[0486]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0488]In another embodiment, the nucleic acid probes may be used in direct
fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends
Genet. 7: 149-154). Although current methods of FISH mapping favor use of
large clones (several kb to several hundred kb; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow performance
of FISH mapping using shorter probes.

[0489]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acid sequences.
Examples include allele-specific amplification (Kazazian (1989) J. Lab.
Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS;
Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation
(Landegren et al. (1988) Science 241:1077-1080), nucleotide extension
reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping
(Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods,
the nucleic acid sequence is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0490]The methods according to the present invention result in plants
having increased yield relative to control plants, as described
hereinbefore. These yield-enhancing traits may also be combined with
other economically advantageous traits, such as further yield-enhancing
traits, tolerance to various stresses, traits modifying various
architectural features and/or biochemical and/or physiological features.

Detailed Description for the SIK Polypeptide

[0491]It has now been found that modulating expression in a plant of a SIK
nucleic acid and/or a SIK polypeptide gives plants having various
improved yield-related traits relative to control plants, wherein
overexpression of a SIK-encoding nucleic acid in a plant gives increased
number of flowers per plant relative to control plants, and wherein the
reduction or substantial elimination of a SIK nucleic acid gives
increased thousand kernel weight, increased harvest index and increased
fill rate relative to corresponding wild type plants.

[0492]Therefore, the invention provides a method for improving
yield-related traits in plants relative to control plants, comprising
modulating expression in a plant of a SIK nucleic acid and/or a SIK
polypeptide, wherein the modulated expression is overexpression of a
SIK-encoding nucleic acid in a plant resulting in an increased number of
flowers per plant relative to control plants, and wherein the modulated
expression is a reduction or substantial elimination of a SIK nucleic
acid resulting in an increased thousand kernel weight (TKW), increased
harvest index (HI) and increased fill rate relative to corresponding wild
type plants.

[0493]The term "modulation" as defined herein is taken to mean a change in
the level of gene expression in comparison to a control plant. In the
case where a SIK-encoding nucleic acid is being used to increase the
number of flowers per plant, the expression level is increased, and in
the case where a SIK nucleic acid is being used to increase TKW, HI or
fill rate, the expression level is decreased. The original, unmodulated
expression may be of any kind of expression of a structural RNA (rRNA,
tRNA) or mRNA with subsequent translation.

[0494]The term "expression" or "gene expression" means the transcription
of a specific gene or specific genes or specific genetic construct. The
term "expression" or "gene expression" in particular means the
transcription of a gene or genes or genetic construct into structural RNA
(rRNA, tRNA) or mRNA with or without subsequent translation of the latter
into a protein. The process includes transcription of DNA and processing
of the resulting mRNA product.

[0496]The improvement may be in one or more of the following: increased
number of flowers per plant, increased seed filling rate (which as
defined herein is the ratio between the number of filled seeds divided by
the total number of seeds; increased harvest index, which as defined
herein is the ratio of the yield of harvestable parts, such as seeds,
divided by the total biomass; and increased thousand kernel weight (TKW),
which as defined herein is derived by extrapolating the number of filled
seeds counted and their total weight. Increased TKW may result from an
increased seed size and/or seed weight, and may also result from an
increase in embryo and/or endosperm size.

[0497]An increase in thousand kernel weight, increased harvest index and
increased fill rate rate occurs whether the plant is under non-stress
conditions or whether the plant is exposed to various stresses compared
to control plants. Plants typically respond to exposure to stress by
growing more slowly. In conditions of severe stress, the plant may even
stop growing altogether. Mild stress on the other hand is defined herein
as being any stress to which a plant is exposed which does not result in
the plant ceasing to grow altogether without the capacity to resume
growth. Mild stress in the sense of the invention leads to a reduction in
the growth of the stressed plants of less than 40%, 35% or 30%,
preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%,
12%, 11% or 10% or less in comparison to the control plant under
non-stress conditions. Due to advances in agricultural practices
(irrigation, fertilization, pesticide treatments) severe stresses are not
often encountered in cultivated crop plants. As a consequence, the
compromised growth induced by mild stress is often an undesirable feature
for agriculture. Mild stresses are the everyday biotic and/or abiotic
(environmental) stresses to which a plant is exposed. Abiotic stresses
may be due to drought or excess water, anaerobic stress, salt stress,
chemical toxicity, oxidative stress and hot, cold or freezing
temperatures. The abiotic stress may be an osmotic stress caused by a
water stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses caused
by pathogens, such as bacteria, viruses, fungi and insects.

[0498]In particular, the methods of the present invention may be performed
under non-stress conditions or under conditions of mild drought to give
plants having increased thousand kernel weight, increased harvest index
and increased fill rate relative to control plants. As reported in Wang
et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of
morphological, physiological, biochemical and molecular changes that
adversely affect plant growth and productivity. Drought, salinity,
extreme temperatures and oxidative stress are known to be interconnected
and may induce growth and cellular damage through similar mechanisms.
Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a
particularly high degree of "cross talk" between drought stress and
high-salinity stress. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption of
homeostasis and ion distribution in the cell. Oxidative stress, which
frequently accompanies high or low temperature, salinity or drought
stress, may cause denaturing of functional and structural proteins. As a
consequence, these diverse environmental stresses often activate similar
cell signalling pathways and cellular responses, such as the production
of stress proteins, up-regulation of anti-oxidants, accumulation of
compatible solutes and growth arrest. The term "non-stress" conditions as
used herein are those environmental conditions that allow optimal growth
of plants. Persons skilled in the art are aware of normal soil conditions
and climatic conditions for a given location.

[0499]Performance of the methods of the invention gives plants grown under
non-stress conditions or under mild drought conditions increased thousand
kernel weight, increased harvest index and increased fill rate relative
to control plants grown under comparable conditions. Therefore, according
to the present invention, there is provided a method for increasing
thousand kernel weight, increased harvest index and increased fill rate
in plants grown under non-stress conditions or under mild drought
conditions, which method comprises increasing expression in a plant of a
nucleic acid encoding a SIK polypeptide.

[0500]Performance of the methods of the invention gives plants grown under
conditions of nutrient deficiency, particularly under conditions of
nitrogen deficiency, increased thousand kernel weight, increased harvest
index and increased fill rate relative to control plants grown under
comparable conditions. Therefore, according to the present invention,
there is provided a method for increasing thousand kernel weight,
increased harvest index and increased fill rate in plants grown under
conditions of nutrient deficiency, which method comprises increasing
expression in a plant of a nucleic acid encoding a SIK polypeptide.
Nutrient deficiency may result from a lack of nutrients such as nitrogen,
phosphates and other phosphorous-containing compounds, potassium,
calcium, cadmium, magnesium, manganese, iron and boron, amongst others.

[0501]The present invention encompasses plants or parts thereof (including
seeds) obtainable by the methods according to the present invention. The
plants or parts thereof comprise a nucleic acid transgene encoding a SIK
polypeptide as defined above.

[0502]According to one aspect of the present invention, a reduction or
substantial elimination of a SIK nucleic acid results in one or more of
an increased thousand kernel weight (TKW), increased harvest index (HI)
and increased fill rate relative to control plants.

[0503]Reference herein to a "SIK nucleic acid" is taken to mean a
polymeric form of a deoxyribonucleotide or a ribonucleotide polymer of
any length, either double- or single-stranded, or analogues thereof, that
has the essential characteristic of a natural ribonucleotide in that it
can hybridise to SIK nucleic acid sequences in a manner similar to
naturally occurring polynucleotides.

[0504]Reference herein to "a reduction or substantial elimination of a SIK
nucleic acid or gene" and to an "endogenous" SIK gene is as described in
the definitions section.

[0505]For the reduction or substantial elimination of expression an
endogenous SIK gene in a plant, a sufficient length of substantially
contiguous nucleotides of a SIK nucleic acid sequence is required. The
stretch of substantially contiguous nucleotides may be derived from any
SIK nucleic acid, preferably a SIK nucleic acid represented by any one of
SEQ ID NO 213 to 225 or any one of the nucleic acid sequences given in
Table 8 or Table 9 below. A SIK nucleic acid sequence encoding a
(functional) polypeptide is not a requirement for the various methods
discussed herein for the reduction or substantial elimination of
expression of an endogenous SIK gene.

[0506]This reduction or substantial elimination may be achieved using
routine tools and techniques, as described above. A preferred method for
the reduction or substantial elimination of expression of a SIK nucleic
acid is by introducing and expressing in a plant a genetic construct into
which the SIK nucleic acid is cloned as an inverted repeat (in part or
completely), separated by a spacer (non-coding DNA).

[0507]According to another aspect of the present invention, overexpression
in a plant of a SIK-encoding nucleic acid results in an increased number
of flowers per plant relative to control plants.

[0508]A preferred method for overexpression of a SIK-encoding nucleic acid
is by introducing and expressing in a plant a nucleic acid encoding a SIK
polypeptide as defined below.

[0509]A "SIK-encoding nucleic acid" encodes a "SIK polypeptide" or "SIK
amino acid sequence" which as defined herein is taken to mean a
polypeptide according to SEQ ID NO: 210 and orthologues and paralogues
thereof as defined herein.

[0510]The SIK polypeptide represented by SEQ ID NO: 210 and orthologues
and paralogues thereof may typically also comprise the following
features.

[0514]The orthologues and paralogues may further comprise one or more
myristoylation sites that could serve to anchor the protein to the
membrane.

Kinase Activity

[0515]Furthermore, the SIK orthologues and paralogues will exhibit kinase
activity of which can readily be determined using routine tools and
techniques. Several assays are available (for example Current Protocols
in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current
Protocols; or online such as http://www.protocol-online.org).

[0516]In brief, the kinase assay generally involves (1) bringing the
kinase protein into contact with a substrate polypeptide containing the
target site to be phosphorylated; (2) allowing phosphorylation of the
target site in an appropriate kinase buffer under appropriate conditions;

[0517](3) separating phosphorylated products from non-phosphorylated
substrate after a suitable reaction period. The presence or absence of
kinase activity is determined by the presence or absence of a
phosphorylated target. In addition, quantitative measurements may be
performed.

[0518]Purified SIK proteins, or cell extracts containing or enriched in
the SIK protein could be used as source for the kinase protein. As a
substrate, small peptides are particularly well suited. The peptide must
comprise one or more serine, threonine, or tyrosine residues in a
phosphorylation site motif. A compilation of phosphorylation sites may be
found in Biochimica et Biophysica Acta 1314, 191-225, (1996). In
addition, the peptide substrates may advantageously have a net positive
charge to facilitate binding to phosphocellulose filters, (allowing to
separate the phosphorylated from non-phosphorylated peptides and to
detect the phosphorylated peptides). If a phosphorylation site motif is
not known, a general tyrosine kinase substrate may be used. For example,
"Src-related peptide" (RRLIEDAEYAARG) is a substrate for many receptor
and non-receptor tyrosine kinases). To determine the kinetic parameters
for phosphorylation of the synthetic peptide, a range of peptide
concentrations is required. For initial reactions, a peptide
concentration of 0.7-1.5 mM may be used.

[0520]A commonly used donor of the phophoryl group is radio-labelled
[gamma-32P]ATP (normally at 0.2 mM final concentration). The amount
of 32P incorporated in the peptides may be determined by measuring
activity on the nitrocellulose dry pads in a scintillation counter.

[0521]Alternatively or additionally, the activity of a SIK orthologue or
paralogue may be assayed by overexpression in a plant of a nucleic acid
encoding a SIK polypeptide under the control of a constitutive promoter
to check for increase the number of flowers per plant relative to control
plants, and also by the reduction or substantial elimination of a SIK
nucleic acid under the control of a constitutive promoter to check for
one or more of an increase in thousand kernel weight (TKW), harvest index
(HI) and fill rate in plants relative to control plants.

[0522]The invention is illustrated by transforming plants with the Oryza
sativa sequence represented by SEQ ID NO: 209, encoding the polypeptide
sequence of SEQ ID NO: 210, however performance of the invention is not
restricted to these sequences. The methods of the invention may
advantageously be performed using any nucleic acid encoding a SIK
polypeptide as defined herein, such as any of the nucleic acid sequences
given in Table 8 and Table 9. The examples of orthologues and paralogues
of SEQ ID NO: 210 given in Table 8 and Table 9 were obtained from The
Institute of Genetic research (TIGR). The % identity given in Table 9 is
on a nucleotide level. The accession number given starting with `TO`
identifies the sequence as found through TIGR. In case of partial nucleic
acids, it would be well within the capabilities of a person skilled in
the art to obtain the full length sequence (or at least a sufficient
length of sequence for performing the methods of the invention).

[0523]Orthologous and paralogous SIK polypeptides may readily be found,
and nucleic acids encoding such orthologues and paralogues would be
useful in performing the methods of the invention.

[0524]Orthologues and paralogues may easily be found by performing a
so-called reciprocal blast search. Typically this involves a first BLAST
involving BLASTing a query sequence (for example, SEQ ID NO: 209 or SEQ
ID NO: 210) against any sequence database, such as the publicly available
NCB! database which may be found at: http://www.ncbi.nlm.nih.gov. BLASTN
or TBLASTX (using standard default values) are generally used when
starting from a nucleotide sequence, and BLASTP or TBLASTN (using
standard default values) when starting from a protein sequence. The BLAST
results may optionally be filtered. The full-length sequences of either
the filtered results or non-filtered results are then BLASTed back
(second BLAST) against sequences from the organism from which the query
sequence is derived (where the query sequence is SEQ ID NO: 209 or SEQ ID
NO: 210, the second BLAST would therefore be against Oryza sequences).
The results of the first and second BLASTs are then compared. A paralogue
is identified if a high-ranking hit from the first blast is from the same
species as from which the query sequence is derived, a BLAST back then
ideally results in the query sequence being among the highest hits; an
orthologue is identified if a high-ranking hit in the first BLAST is not
from the same species as from which the query sequence is derived, and
preferably results upon BLAST back in the query sequence being among the
highest hits.

[0525]High-ranking hits are those having a low E-value. The lower the
E-value, the more significant the score (or in other words the lower the
chance that the hit was found by chance). Computation of the E-value is
well known in the art. In addition to E-values, comparisons are also
scored by percentage identity. Percentage identity refers to the number
of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length.
Preferably the score is greater than 50, more preferably greater than
100; and preferably the E-value is less than e-5, more preferably less
than e-6. In the case of large families, ClustalW may be used, followed
by a neighbour joining tree, to help visualize clustering of related
genes and to identify orthologues and paralogues.

[0526]Orthologues and paralogues may also be identified using the BLAST
procedure described below. Homologues (or homologous proteins,
encompassing orthologues and paralogues) may readily be identified using
routine techniques well known in the art, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in
the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP
uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:
443-453) to find the alignment of two complete sequences that maximizes
the number of matches and minimizes the number of gaps. The BLAST
algorithm (Altschul et al. (1990) J Mol Biol 215: 403-410) calculates
percent sequence identity and performs a statistical analysis of the
similarity between the two sequences. The software for performing BLAST
analysis is publicly available through the National Centre for
Biotechnology Information. Homologues may readily be identified using,
for example, the ClustalW multiple sequence alignment algorithm (version
1.83), with the default pairwise alignment parameters, and a scoring
method in percentage. Global percentages of similarity and identity may
also be determined using one of the methods available in the MatGAT
software package (Campanella et al., BMC Bioinformatics. 4, 29, 2003).
Minor manual editing may be performed to optimise alignment between
conserved motifs, as would be apparent to a person skilled in the art.
Furthermore, instead of using full-length sequences for the
identification of homologues, specific domains may be used as well. The
sequence identity values, which are indicated below as a percentage were
determined over the entire SIK nucleic acid or amino acid sequence using
the programs mentioned above using the default parameters.

[0528]Alternatively, the sequence identity among homologues may be
determined using a specific domain. Any given domain may be identified
and delineated using the databases and tools for protein identification
listed above, and/or methods for the alignment of sequences for
comparison. In some instances, default parameters may be adjusted to
modify the stringency of the search. For example using BLAST, the
statistical significance threshold (called "expect" value) for reporting
matches against database sequences may be increased to show less
stringent matches. In this way, short nearly exact matches may be
identified.

[0530]Also useful in the methods of the invention are nucleic acids
encoding homologues of a SIK polypeptide represented by SEQ ID NO: 210 or
orthologues or paralogues thereof as defined above.

[0531]Also useful in the methods of the invention are nucleic acids
encoding derivatives of SEQ ID NO: 210 or nucleic acids encoding
derivatives of orthologues, paralogues or homologues of SEQ ID NO: 210.

[0532]Nucleic acids encoding SIK polypeptides defined herein need not be
full-length nucleic acids, since performance of the methods of the
invention does not rely on the use of full length nucleic acid sequences.
Examples of nucleic acids suitable for use in performing the methods of
the invention include the nucleic acid sequences given in Table 8 and
Table 9, but are not limited to those sequences. Nucleic acid variants
may also be useful in practising the methods of the invention. Examples
of such nucleic acid variants include portions of nucleic acids encoding
a SIK polypeptide as defined herein, splice variants of nucleic acids
encoding a SIK polypeptide as defined herein, allelic variants of nucleic
acids encoding a SIK polypeptide as defined herein and variants of
nucleic acids encoding a SIK polypeptide as defined herein that are
obtained by gene shuffling. The terms portion, splice variant, allelic
variant and gene shuffling are as described herein.

[0533]According to the present invention, there is provided a method for
increasing the number of flowers per plant relative to control plants,
comprising introducing and expressing in a plant a portion of any one of
the nucleic acid sequences given in Table 8 or Table 9, or a portion of a
nucleic acid encoding an orthologue, paralogue or homologue of any of the
amino acid sequences given in Table 8 or Table 9.

[0534]Portions useful in the methods of the invention, encode a
polypeptide having substantially the same biological activity as the SIK
polypeptide represented by any of the amino acid sequences given in Table
8 or Table 9. Preferably, the portion is a portion of any one of the
nucleic acids given in Table 8 or Table 9, or a portion of any one of the
nucleic acids sequences represented by SEQ ID NO: 213 to 225. The portion
is typically at least 300 consecutive nucleotides in length, preferably
at least 400 consecutive nucleotides in length, more preferably at least
500 consecutive nucleotides in length, the consecutive nucleotides being
of any one of the nucleic acid sequences given in Table 8 or Table 9 or
any one of the nucleic acids sequences represented by SEQ ID NO: 213 to
225. Most preferably the portion is a portion of the nucleic acid of SEQ
ID NO: 209. Preferably, the portion encodes an amino acid sequence
comprising any one or more of an ATP-binding region, a serine threonine
kinase active site signature, an N-terminal serine-rich domain, one or
more myristoylation sites and kinase activity.

[0535]A portion of a nucleic acid encoding a SIK polypeptide as defined
herein may be prepared, for example, by making one or more deletions to
the nucleic acid. The portions may be used in isolated form or they may
be fused to other coding (or non coding) sequences in order to, for
example, produce a protein that combines several activities. When fused
to other coding sequences, the resultant polypeptide produced upon
translation may be bigger than that predicted for the portion.

[0536]Another nucleic acid variant useful in the methods of the invention
is a nucleic acid capable of hybridising, under reduced stringency
conditions, preferably under stringent conditions, with a nucleic acid
encoding a SIK polypeptide as defined herein, or with a portion as
defined herein.

[0537]Hybridising sequences useful in the methods of the invention, encode
a polypeptide having substantially the same biological activity as the
SIK polypeptide represented by any of the amino acid sequences given in
Table 8 or Table 9. The hybridising sequence is typically at least 300
consecutive nucleotides in length, preferably at least 400 consecutive
nucleotides in length, more preferably at least 500 consecutive
nucleotides in length, the consecutive nucleotides being of any one of
the nucleic acid sequences given in Table 8 or Table 9. Preferably, the
hybridising sequence is one that is capable of hybridising to any of the
nucleic acids given in Table 8 or Table 9, or to a portion of any of
these sequences, a portion being as defined above. Most preferably, the
hybridising sequence is capable of hybridising to a nucleic acid as
represented by SEQ ID NO: 209 or to a portion thereof. Preferably, the
hybridising sequence encodes an amino acid sequence comprising any one or
more of an ATP-binding region, a serine threonine kinase active site
signature, an N-terminal serine-rich domain, one or more myristoylation
sites and kinase activity.

[0538]According to the present invention, there is provided a method for
increasing the number of flowers per plant, comprising introducing and
expressing in a plant a nucleic acid capable of hybridizing to any one of
the nucleic acids given in Table 8 or Table 9 or any one of the nucleic
acids sequences represented by SEQ ID NO: 213 to 225, or comprising
introducing and expressing in a plant a nucleic acid capable of
hybridising to a nucleic acid encoding an orthologue, paralogue or
homologue of any of the nucleic acid sequences given in Table 1 or Table
2 or any one of the nucleic acids sequences represented by SEQ ID NO: 213
to 225.

[0539]Another nucleic acid variant useful in the methods of the invention
is a splice variant encoding a SIK polypeptide as defined hereinabove,
the term "splice variant" being as defined above.

[0540]According to the present invention, there is provided a method for
increasing the number of flowers per plant, comprising introducing and
expressing in a plant a splice variant of any one of the nucleic acid
sequences given in Table 8 or Table 9 or a splice variant of any one of
the nucleic acids represented by SEQ ID NO: 213 to 225, or a splice
variant of a nucleic acid encoding an orthologue, paralogue or homologue
of any of the amino acid sequences given in Table 8 or Table 9 or any one
of the nucleic acids sequences represented by SEQ ID NO: 213 to 225.

[0541]Preferred splice variants are splice variants of a nucleic acid
represented by SEQ ID NO: 209 or a splice variant encoding an orthologue
or paralogue of SEQ ID NO: 210. Preferably, the amino acid encoded by the
splice variant comprises any one or more of an ATP-binding region, a
serine threonine kinase active site signature, an N-terminal serine-rich
domain, one or more myristoylation sites and kinase activity.

[0542]Another nucleic acid variant useful in performing the methods of the
invention is an allelic variant of a nucleic acid encoding a SIK
polypeptide as defined hereinabove.

[0543]According to the present invention, there is provided a method for
increasing the number of flowers per plant, comprising introducing and
expressing in a plant an allelic variant of any one of the nucleic acids
given in Table 8 or Table 9 or of any one of the nucleic acid sequences
represented by SEQ ID NO: 213 to 225, or comprising introducing and
expressing in a plant an allelic variant of a nucleic acid encoding an
orthologue, paralogue or homologue of any of the amino acid sequences
given in Table 8 or Table 9 or of any one of the nucleic acids sequences
represented by SEQ ID NO: 213 to 225.

[0544]Preferably, the allelic variant is an allelic variant of SEQ ID NO:
209 or an allelic variant of a nucleic acid encoding an orthologue or
paralogue or homologue of SEQ ID NO: 210. Preferably, the amino acid
encoded by the allelic variant comprises any one or more of an
ATP-binding region, a serine threonine kinase active site signature, an
N-terminal serine-rich domain, one or more myristoylation sites and
kinase activity.

[0545]A further nucleic acid variant useful in the methods of the
invention is a nucleic acid variant obtained by gene shuffling. Gene
shuffling or directed evolution may also be used to generate variants of
nucleic acids encoding SIK polypeptides.

[0546]According to the present invention, there is provided a method for
increasing the number of flowers per plant, comprising introducing and
expressing in a plant a variant of any one of the nucleic acid sequences
given in Table Table 8 or Table 9, or of any one of the nucleic acids
sequences represented by SEQ ID NO: 213 to 225, or comprising introducing
and expressing in a plant a variant of a nucleic acid encoding an
orthologue, paralogue or homologue of any of the amino acid sequences
given in Table 8 or Table 9 or of any one of the nucleic acids sequences
represented by SEQ ID NO: 213 to 225, which variant nucleic acid is
obtained by gene shuffling. Preferably, the variant nucleic acid obtained
by gene shuffling encodes an amino acid comprising any one or more of an
ATP-binding region, a serine threonine kinase active site signature, an
N-terminal serine-rich domain, one or more myristoylation sites and
kinase activity.

[0547]Furthermore, nucleic acid variants may also be obtained by
site-directed mutagenesis. Several methods are available to achieve
site-directed mutagenesis, the most common being PCR based methods
(current protocols in molecular biology. Wiley Eds.

[0548]SIK nucleic acids encoding SIK-like polypeptides may be derived from
any natural or artificial source. The SIK nucleic acid may be modified
from its native form in composition and/or genomic environment through
deliberate human manipulation. Preferably the SIK-encoding nucleic acid
is from a plant, further preferably from a monocotyledonous plant, more
preferably from the family of Poaceae, most preferably the nucleic acid
is from Oryza sativa.

[0549]Any reference herein to a SIK polypeptide is therefore taken to mean
a SIK polypeptide as defined above. Any SIK nucleic acid encoding such a
SIK polypeptide is suitable for use in performing the methods of the
invention to increase the number of flowers per plant.

[0550]The present invention also encompasses plants or parts thereof
(including seeds) obtainable by the methods according to the present
invention. The plants or parts thereof comprise a nucleic acid transgene
encoding a SIK polypeptide as defined above.

[0551]The invention also provides genetic constructs and vectors to
facilitate introduction and/or expression of the SIK nucleic acid
sequences useful in the methods according to the invention, in a plant.

[0552]Therefore, there is provided a gene construct comprising:
[0553](i) a SIK nucleic acid as defined hereinabove or a SIK-encoding
nucleic acid; [0554](ii) one or more control sequences operably linked to
the SIK nucleic acid of (i).

[0555]A preferred construct in the case of reduction or substantial
elimination of a SIK nucleic acid to give increased thousand kernel
weight, increased harvest index and increased fill rate is one comprising
an inverted repeat of a SIK nucleic acid, preferably capable of forming a
hairpin structure, which inverted repeat is under the control of a
constitutive promoter.

[0556]Constructs useful in the methods according to the present invention
may be constructed using recombinant DNA technology well known to persons
skilled in the art. The gene constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for transcribing of the gene of interest in the
transformed cells. The invention therefore provides use of a gene
construct as defined hereinabove in the methods of the invention.

[0557]Plants are transformed with a vector comprising the sequence of
interest. The skilled artisan is well aware of the genetic elements that
must be present on the vector in order to successfully transform, select
and propagate host cells containing the sequence of interest. The
sequence of interest is operably linked to one or more control sequences
(at least to a promoter). The terms "regulatory element", "control
sequence" and "promoter" are all used interchangeably herein and are
defined above.

[0558]Advantageously, any type of promoter may be used in the methods o
the present invention. The term "promoter" refers to a nucleic acid
control sequence located upstream from the transcriptional start of a
gene and which is involved in recognising and binding of RNA polymerase
and other proteins, thereby directing transcription of an operably linked
nucleic acid. The promoter may be a constitutive promoter. Alternatively,
the promoter may be an inducible promoter. Additionally or alternatively,
the promoter may be a tissue-specific promoter. Promoters able to
initiate transcription in certain tissues only are referred to herein as
"tissue-specific", similarly, promoters able to initiate transcription in
certain cells only are referred to herein as "cell-specific".

[0559]Preferably, the nucleic acid sequence is operably linked to a
constitutive promoter. Preferably the promoter is derived from a plant,
more preferably the promoter is from a monocotyledonous plant if a
monocotyledonous plant is to be transformed. Further preferably, the
constitutive promoter is a GOS2 promoter that is represented by a nucleic
acid sequence substantially similar to SEQ ID NO: 56 or 226. Most
preferably the GOS2 promoter is as represented by SEQ ID NO: 56 or 226.
It should be clear that the applicability of the present invention is not
restricted to the SIK nucleic acid sequence as represented by SEQ ID NO:
209, nor is the applicability of the invention restricted to expression
of a SIK nucleic acid sequence when driven by a GOS2 promoter. Examples
of other constitutive promoters that may also be used to drive expression
of a SIK nucleic acid sequence are shown above.

[0560]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0561]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-ori and colE1.

[0562]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). Therefore, the genetic construct
may optionally comprise a selectable marker gene. Selectable markers are
described in more detail in the "definitions" section herein. The marker
genes may be removed or excised from the transgenic cell once they are no
longer needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section.

[0563]The expression of a SIK nucleic acid or SIK polypeptide may also be
modulated by introducing a genetic modification, within the locus of a
SIK gene. The locus of a gene as defined herein is taken to mean a
genomic region, which includes the gene of interest and 10 kb up- or down
stream of the coding region.

[0564]The genetic modification may be introduced, for example, by any one
(or more) of the following methods: T-DNA tagging, TILLING and homologous
recombination. Following introduction of the genetic modification, there
follows a step of selecting for suitable modulated expression.

[0565]The methods of the invention are advantageously applicable to any
plant. Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0567]The present invention also encompasses plants obtainable by the
methods according to the present invention. The present invention
therefore provides plants, plant parts or plant cells thereof obtainable
by the method according to the present invention, which plants or parts
or cells thereof comprise a SIK nucleic acid transgene (which may encode
a SIK polypeptide as defined above).

[0568]The invention furthermore provides a method for the production of
transgenic plants having the various improved yield-related traits
mentioned above, comprising introduction and expression in a plant SIK
nucleic acid as defined.

[0569]Host plants for the nucleic acids or the vector used in the method
according to the invention, the expression cassette or construct or
vector are, in principle, advantageously all plants, which are capable of
synthesizing the polypeptides used in the inventive method.

[0570]More specifically, the present invention provides a method for the
production of transgenic plants having various improved yield-related
traits relative to control plants, which method comprises: [0571](i)
introducing and expressing a SIK nucleic acid in a plant cell; and
[0572](ii) cultivating the plant cell under conditions promoting plant
growth and development; [0573](iii) obtaining plants having increased
number of flowers per plant.

[0574]Also provided is a method for the production of transgenic plants
having various improved yield-related traits relative to control plants,
which method comprises: [0575](i) introducing into a plant cell a
construct for downregulating SIK gene expression; and [0576](ii)
cultivating the plant cell under conditions promoting plant growth and
development; [0577](iii) obtaining plants having one or more of increased
thousand kernel weight, incraesd harvest index and increased fill rate.

[0578]The nucleic acid may be introduced directly into a plant cell or
into the plant itself (including introduction into a tissue, organ or any
other part of a plant). According to a preferred feature of the present
invention, the nucleic acid is preferably introduced into a plant by
transformation.

[0579]Further preferably the construct for downregulating SIK gene
expression and introduced into the plant cell or plant comprise an
inverted repeat of the SIK nucleic acid or a part thereof.

[0580]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant.

[0581]As mentioned Agrobacteria transformed with an expression vector
according to the invention may also be used in the manner known per se
for the transformation of plants such as experimental plants like
Arabidopsis or crop plants, such as, for example, cereals, maize, oats,
rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower,
flax, hemp, potato, tobacco, tomato, carrot, bell peppers, oilseed rape,
tapioca, cassaya, arrow root, tagetes, alfalfa, lettuce and the various
tree, nut, and grapevine species, in particular oil-containing crop
plants such as soya, peanut, castor-oil plant, sunflower, maize, cotton,
flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius)
or cocoa beans, for example by bathing scarred leaves or leaf segments in
an agrobacterial solution and subsequently growing them in suitable
media.

[0582]The genetically modified plant cells can be regenerated via all
methods with which the skilled worker is familiar. Suitable methods can
be found in the abovementioned publications by S.D. Kung and R. Wu,
Potrykus or Hofgen and Willmitzer.

[0583]To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0584]Following DNA transfer and regeneration, putatively transformed
plants may be evaluated, for instance using Southern analysis, for the
presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels may be
monitored using Northern and/or Western analysis, or quantitative PCR,
all techniques being well known to persons having ordinary skill in the
art.

[0585]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
to give homozygous second generation (or T2) transformants, and the T2
plants further propagated through classical breeding techniques.

[0586]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0587]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention.

[0588]The invention also includes host cells containing an isolated SIK
nucleic acid as defined hereinabove. Preferred host cells according to
the invention are plant cells. Host plants for the nucleic acids or the
vector used in the method according to the invention, the expression
cassette or construct or vector are, in principle, advantageously all
plants, which are capable of synthesizing the polypeptides used in the
inventive method.

[0589]The invention also extends to harvestable parts of a plant such as,
but not limited to seeds, leaves, fruits, flowers, stems, rhizomes,
tubers and bulbs. The invention furthermore relates to products derived,
preferably directly derived, from a harvestable part of such a plant,
such as dry pellets or powders, oil, fat and fatty acids, starch or
proteins.

[0590]The present invention also encompasses use of SIK nucleic acids and
SIK polypeptides in improving various yield-related traits as mentioned
above.

[0591]Nucleic acids encoding SIK polypeptides may find use in breeding
programmes in which a DNA marker is identified which may be genetically
linked to a SIK gene. The nucleic acids/genes may be used to define a
molecular marker. This DNA marker may then be used in breeding programmes
to select plants having increased yield as defined hereinabove in the
methods of the invention.

[0592]Allelic variants of a SIK nucleic acid/gene may also find use in
marker-assisted breeding programmes. Such breeding programmes sometimes
require introduction of allelic variation by mutagenic treatment of the
plants, using for example EMS mutagenesis; alternatively, the programme
may start with a collection of allelic variants of so called "natural"
origin caused unintentionally. Identification of allelic variants then
takes place, for example, by PCR. This is followed by a step for
selection of superior allelic variants of the sequence in question and
which give increased yield. Selection is typically carried out by
monitoring growth performance of plants containing different allelic
variants of the sequence in question. Growth performance may be monitored
in a greenhouse or in the field. Further optional steps include crossing
plants in which the superior allelic variant was identified with another
plant. This could be used, for example, to make a combination of
interesting phenotypic features.

[0593]A SIK nucleic acid may also be used as probes for genetically and
physically mapping the genes that they are a part of, and as markers for
traits linked to those genes. Such information may be useful in plant
breeding in order to develop lines with desired phenotypes. Such use of
SIK nucleic acids requires only a nucleic acid sequence of at least 15
nucleotides in length. The SIK nucleic acids may be used as restriction
fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J,
Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual)
of restriction-digested plant genomic DNA may be probed with the SIK
nucleic acids. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander et al.
(1987) Genomics 1: 174-181) in order to construct a genetic map. In
addition, the nucleic acids may be used to probe Southern blots
containing restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic cross.
Segregation of the DNA polymorphisms is noted and used to calculate the
position of the SIK nucleic acid in the genetic map previously obtained
using this population (Botstein et al. (1980) Am. J. Hum. Genet.
32:314-331).

[0594]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bernatzky and Tanksley (Plant Mol. Biol.
Reporter 4: 37-41, 1986). Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0596]In another embodiment, the nucleic acid probes may be used in direct
fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favour use of
large clones (several kb to several hundred kb; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow performance
of FISH mapping using shorter probes.

[0597]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acids. Examples
include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med
11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et
al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and
Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the
sequence of a nucleic acid is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0598]The methods according to the present invention result in plants
having altered yield-related traits, as described hereinbefore. These
traits may also be combined with other economically advantageous traits,
such as further yield-enhancing traits, tolerance to other abiotic and
biotic stresses, traits modifying various architectural features and/or
biochemical and/or physiological features.

Detailed Description for the Class II HD-Zip Transcription Factors

[0599]It has now been found that nucleic acids encoding Class II HD-Zip
transcription factors are useful in modifying the content of storage
compounds in seeds. The present invention therefore provides a method for
modifying the content of storage compounds in seeds relative to control
plants by modulating expression in a plant of a nucleic acid encoding a
Class II HD-Zip transcription factor. The present invention also provides
nucleic acid sequences and constructs useful in performing such methods.
The invention further provides seeds having a modified content of storage
compounds relative to control plants, which seeds have modulated
expression of a nucleic acid encoding a Class II HD-Zip transcription
factor.

[0600]The present invention provides a method for modifying the content of
storage compounds in seeds relative to control plants, comprising
modulating expression in a plant of a nucleic acid encoding a Class II
HD-Zip transcription factor.

[0601]A preferred method for modulating (preferably, increasing)
expression of a nucleic acid encoding a Class II HD-Zip transcription
factor is by introducing and expressing in a plant a nucleic acid
encoding a Class II HD-Zip transcription factor as will now be defined.

[0602]A "Class II HD-Zip transcription factor" is taken to mean a
polypeptide comprising the following: (i) a homeodomain box; (ii) a
leucine zipper; and (iii) Motifs I, II and III given below (in any
order).

Motif I (SEQ ID NO: 279)

[0603]RKKLRL, or Motif I with one or more conservative amino acid
substitution at any position, and/or Motif I with one or two
non-conservative change(s) at any position; and

[0604]Motif II (SEQ ID NO: 280)

[0605]TKLKQTEVDCEFLRRCCENLTEEN, or Motif II with one or more conservative
amino acid substitution at any position, and/or a motif having in
increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or more sequence identity to Motif II; and

Motif III (SEQ ID NO: 281)

[0606]TLTMCPSCER, or Motif III with one or more conservative amino acid
substitution at any position, and/or Motif III with one, two or three
non-conservative change(s) at any position.

[0607]Any reference herein to a "nucleic acid encoding a Class II HD-Zip
transcription factor" or to a Class II HD-Zip-encoding nucleic acid" is
taken to mean a nucleic acid encoding a Class II HD-Zip transcription
factor as defined hereinabove, such nucleic acids being useful in
performing the methods of the invention.

[0608]As mentioned, a preferred method for modulating (preferably,
increasing) expression of a nucleic acid encoding a Class II HD-Zip
transcription factor is by introducing and expressing in a plant a
nucleic acid encoding a Class II HD-Zip transcription factor.

[0610]However, performance of the invention is not restricted to these
sequences; the methods of the invention may advantageously be performed
using any Class II HD-Zip transcription factor-encoding nucleic acid or
Class II HD-Zip transcription factor as defined herein.

[0611]For example, nucleic acids encoding orthologues or paralogues of an
amino acid sequence represented by SEQ ID NO: 230 may be useful in
performing the methods of the invention. Examples of such orthologues and
paralogues are provided in Table A of Example 1.

[0612]Orthologues and paralogues encompass evolutionary concepts used to
describe the ancestral relationships of genes. Paralogues are genes
within the same species that have originated through duplication of an
ancestral gene and orthologues are genes from different organisms that
have originated through speciation.

[0613]Orthologues and paralogues may easily be found by performing a
so-called reciprocal blast search. Typically this involves a first BLAST
involving BLASTing a query sequence (for example, SEQ ID NO: 229 or SEQ
ID NO: 230) against any sequence database, such as the publicly available
NCB! database which may be found at: http://www.ncbi.nlm.nih.gov. BLASTN
or TBLASTX (using standard default values) are generally used when
starting from a nucleotide sequence, and BLASTP or TBLASTN (using
standard default values) when starting from a protein sequence. The BLAST
results may optionally be filtered. The full-length sequences of either
the filtered results or non-filtered results are then BLASTed back
(second BLAST) against sequences from the organism from which the query
sequence is derived (where the query sequence is SEQ ID NO: 229 or SEQ ID
NO: 230, the second BLAST would therefore be against Oryza sequences).
The results of the first and second BLASTs are then compared. A paralogue
is identified if a high-ranking hit from the first blast is from the same
species as from which the query sequence is derived, a BLAST back then
ideally results in the query sequence being among the highest hits; an
orthologue is identified if a high-ranking hit in the first BLAST is not
from the same species as from which the query sequence is derived, and
preferably results upon BLAST back in the query sequence being among the
highest hits. High-ranking hits are those having a low E-value. The lower
the E-value, the more significant the score (or in other words the lower
the chance that the hit was found by chance). Computation of the E-value
is well known in the art. In addition to E-values, comparisons are also
scored by percentage identity. Percentage identity refers to the number
of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length. In the
case of large families, ClustalW may be used, followed by a neighbour
joining tree, to help visualize clustering of related genes and to
identify orthologues and paralogues.

[0614]Nucleic acids encoding homologues of an amino acid sequence
represented by SEQ ID NO: 230, or nucleic acids encoding homologues of
any of the amino acid sequences given in Table H, may also be useful in
performing the methods of the invention.

[0615]Also useful in the methods of the invention are nucleic acids
encoding derivatives of any one of the amino acids given in Table H or
derivatives of orthologues or paralogues of any of the amino acid
sequences given in Table H. "Derivatives" include peptides,
oligopeptides, polypeptides which may, compared to the amino acid
sequence of the naturally-occurring form of the protein, such as the one
presented in SEQ ID NO: 230, comprise substitutions of amino acids with
non-naturally occurring amino acid residues, or additions of
non-naturally occurring amino acid residues.

[0616]Homologues (or homologous proteins, encompassing orthologues and
paralogues) may readily be identified using routine techniques well known
in the art, such as by sequence alignment. Methods for the alignment of
sequences for comparison are well known in the art, such methods include
GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of
Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the
alignment of two complete sequences that maximizes the number of matches
and minimizes the number of gaps. The BLAST algorithm (Altschul et al.
(1990) J Mol Biol 215: 403-410) calculates percent sequence identity and
performs a statistical analysis of the similarity between the two
sequences. The software for performing BLAST analysis is publicly
available through the National Centre for Biotechnology Information.
Homologues may readily be identified using, for example, the ClustalW
multiple sequence alignment algorithm (version 1.83), with the default
pairwise alignment parameters, and a scoring method in percentage. Global
percentages of similarity and identity may also be determined using one
of the methods available in the MatGAT software package (Campanella et
al., BMC Bioinformatics. 4, 29, 2003). Minor manual editing may be
performed to optimise alignment between conserved motifs, as would be
apparent to a person skilled in the art. Furthermore, instead of using
full-length sequences for the identification of homologues, specific
domains may be used as well.

[0617]The sequence identity values, which are indicated below as a
percentage were determined over the entire nucleic acid or amino acid
sequence using ClustalW and default parameters.

[0619]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of a nucleic acid
encoding a Class II HD-Zip transcription factor represented by SEQ ID NO:
2 or an orthologue, paralogue or homologue thereof.

[0620]A preferred method for modulating (preferably, increasing)
expression of a nucleic acid encoding a Class II HD-Zip transcription
factor is by introducing and expressing in a plant a nucleic acid
encoding a Class II HD-Zip transcription factor represented by SEQ ID NO:
230 or an orthologue, paralogue or homologue thereof.

[0621]The orthologues, paralogues and homologues described above fall
under the definition of a Class II HD-Zip transcription factor, i.e.
meaning that the orthologues, paralogues and homologues are polypeptides
comprising the following: (i) a homeodomain box; (ii) a leucine zipper;
and (iii) Motifs I, II and III described herein.

[0623]Nucleic acids useful in the methods of the invention need not be
full-length, since performance of the methods of the invention does not
rely on the use of full length nucleic acids. Examples include portions
of the nucleic acid sequence represented by SEQ ID NO: 229 or portions of
any one of the nucleic acid sequences given in Table H.

[0624]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of a portion of a
nucleic acid sequence represented by SEQ ID NO: 229, or comprising
modulating expression in a plant of a portion of any one of the nucleic
acid sequences given in Table H, or comprising modulating expression in a
plant of a portion of a nucleic acid sequence encoding an orthologue,
paralogue or homologue of any of the amino acid sequences given in Table
H.

[0625]A preferred method for modulating (preferably, increasing)
expression in a plant of such a portion is by introducing and expressing
in a plant a portion of a nucleic acid sequence given in Table H, or
comprising introducing and expressing in a plant a portion of a nucleic
acid sequence encoding an orthologue, paralogue or homologue of any of
the polypeptide sequences given in Table H.

[0626]Portions useful in the methods of the invention, include portions of
sufficient length to encode a polypeptide falling under the definition of
a Class II HD-Zip transcription factor, i.e. meaning that the polypeptide
comprises the following: (i) a homeodomain box; (ii) a leucine zipper;
and (iii) Motifs I, II and III described herein. Furthermore, such
portions have substantially the same biological activity as Class II
HD-Zip transcription factors.

[0627]Preferably, the portion is at least 500 consecutive nucleotides in
length, preferably at least 750 consecutive nucleotides in length, more
preferably at least 1,250 consecutive nucleotides in length, the
consecutive nucleotides being of SEQ ID NO: 229, or of any one of the
nucleic acid sequences given in Table H, or of any nucleic acid encoding
an orthologue, paralogue or homologue of any one of the amino acid
sequences given in Table H. Most preferably the portion is a portion of
the nucleic acid of SEQ ID NO: 229.

[0628]A portion as defined herein may be prepared, for example, by making
one or more deletions to the nucleic acid in question. The portions may
be used in isolated form or they may be fused to other coding (or non
coding) sequences in order to, for example, produce a protein that
combines several activities. When fused to other coding sequences, the
resultant polypeptide produced upon translation may be bigger than that
predicted for the portion.

[0629]Another nucleic acid useful in the methods of the invention is a
nucleic acid capable of hybridising, under reduced stringency conditions,
preferably under medium stringency conditions, more preferably under
stringent conditions, with a nucleic acid represented by SEQ ID NO: 229,
or capable of hybridising, under reduced stringency conditions,
preferably under medium stringency conditions, more preferably under
stringent conditions, with a nucleic acid sequence given in Table H, or
capable of hybridising under reduced stringency conditions, preferably
under medium stringency conditions, more preferably under stringent
conditions with a nucleic acid encoding an orthologue, paralogue or
homologue of a polypeptide sequence given in Table H.

[0630]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of a nucleic acid
capable of hybridising to a nucleic acid represented by SEQ ID NO: 229,
or comprising modulating expression in a plant of a nucleic acid capable
of hybridising to a nucleic acid sequence given in Table H, or comprising
modulating expression in a plant of a nucleic acid capable of hybridising
to a nucleic acid sequence encoding an orthologue, paralogue or homologue
of any of the polypeptide sequences given in Table H. Most preferably the
hybridising sequence is capable of hybridising to a nucleic acid as
represented by SEQ ID NO: 229. The hybridising sequence is preferably
capable of hybridising under reduced stringency conditions, preferably
under medium stringency conditions, more preferably under stringent
conditions.

[0631]A preferred method for modulating (preferably, increasing)
expression in a plant of such a hybridising sequence is by introducing
and expressing in a plant a nucleic acid capable of hybridising to a
nucleic acid sequence given in Table H, or comprising introducing and
expressing in a plant of a nucleic acid sequence capable of hybridising
to a nucleic acid sequence encoding an orthologue, paralogue or homologue
of any one of the amino acid sequences given in Table H. Most preferably
the hybridising sequence is capable of hybridising to a nucleic acid as
represented by SEQ ID NO: 229. The hybridising sequence is preferably
capable of hybridising under reduced stringency conditions, preferably
under medium stringency conditions, more preferably under stringent
conditions.

[0632]Hybridising sequences useful in the methods of the invention,
include nucleic acids of sufficient length to encode a polypeptide
falling under the definition of a Class II HD-Zip transcription factor,
i.e. meaning that the polypeptide comprises the following: (i) a
homeodomain box; (ii) a leucine zipper; and (iii) Motifs I, II and III
described herein. Furthermore, such hybridising sequences have
substantially the same biological activity as the Class II HD-Zip
transcription factors.

[0633]The hybridising sequence is typically at least 500 consecutive
nucleotides in length, preferably at least 750 consecutive nucleotides in
length, more preferably at least 1,250 consecutive nucleotides in length,
the consecutive nucleotides being of any nucleic acid capable of
hybridising to a nucleic acid sequence given in Table H, or of any
nucleic acid encoding an orthologue, paralogue or homologue of any one of
the amino acid sequences given in Table H. Most preferably the
consecutive nucleotides are of a nucleic acid capable of hybridising to a
nucleic acid represented by SEQ ID NO: 229 or to a portion thereof.

[0634]Another nucleic acid useful in the methods of the invention is a
splice variant of SEQ ID NO: 229, or a splice variant of any of the
nucleic acid sequences given in Table H, or a splice variant of a nucleic
acid encoding an orthologue, paralogue or homologue of any one of the
amino acid sequences given in Table H.

[0635]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of a splice variant
of a nucleic acid represented by SEQ ID NO: 229, or comprising modulating
expression in a plant of a splice variant of nucleic acid sequence given
in Table H, or comprising modulating expression in a plant of a splice
variant of a nucleic acid sequence encoding an orthologue, paralogue or
homologue of any one of the amino acid sequences given in Table H.

[0636]A preferred method for modulating (preferably, increasing)
expression in a plant of such a splice variant is by introducing and
expressing in a plant a splice variant of a nucleic acid sequence given
in Table H, or comprising introducing and expressing in a plant a splice
variant of a nucleic acid sequence encoding an orthologue, paralogue or
homologue of any one of the amino acid sequences given in Table H.

[0637]Splice variants useful in the methods of the invention, include
nucleic acids of sufficient length to encode a polypeptide falling under
the definition of a Class II HD-Zip transcription factor, i.e. meaning
that the polypeptide comprises the following: (i) a homeodomain box; (ii)
a leucine zipper; and (iii) Motifs I, II and III described herein.
Furthermore, such splice variants have substantially the same biological
activity as the Class II HD-Zip transcription factors.

[0638]Another nucleic acid useful in performing the methods of the
invention is an allelic variant of SEQ ID NO: 229, or an allelic variant
of any of the nucleic acid sequences given in Table H, or an allelic
variant of a nucleic acid encoding an orthologue, paralogue or homologue
of any one of the amino acid sequences given in Table H. Allelic variants
exist in nature, and encompassed within the methods of the present
invention is the use of these natural alleles.

[0639]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of an allelic variant
of a nucleic acid represented by SEQ ID NO: 229, or comprising modulating
expression in a plant of an allelic variant of nucleic acid sequence
given in Table H, or comprising modulating expression in a plant of an
allelic variant of a nucleic acid sequence encoding an orthologue,
paralogue or homologue of any one of the amino acid sequences given in
Table H.

[0640]A preferred method for modulating (preferably, increasing)
expression in a plant of such an allelic variant is by introducing and
expressing in a plant an allelic variant of a nucleic acid sequence given
in Table H, or comprising introducing and expressing in a plant an
allelic variant of a nucleic acid sequence encoding an orthologue,
paralogue or homologue of any one of the amino acid sequences given in
Table H.

[0641]Allelic variants useful in the methods of the invention, include
nucleic acids of sufficient length to encode a polypeptide falling under
the definition of a Class II HD-Zip transcription factor, i.e. meaning
that the polypeptide comprises the following: (i) a homeodomain box; (ii)
a leucine zipper; and (iii) Motifs I, II and III described herein.
Furthermore, such allelic variants have substantially the same biological
activity as the Class II HD-Zip transcription factors.

[0642]A further nucleic acid useful in the methods of the invention is a
nucleic acid obtained by gene shuffling. Gene shuffling or directed
evolution may also be used to generate variants of any one of the nucleic
acids given in Table H, or variants of nucleic acids encoding
orthologues, paralogues or homologues of any one of the amino acid
sequences given in Table H.

[0643]According to the present invention, there is provided a method for
modifying the content of storage compounds in seeds relative to control
plants, comprising modulating expression in a plant of a variant of a
nucleic acid represented by SEQ ID NO: 229, or comprising modulating
expression in a plant of a variant of any one of the nucleic acid
sequences given in Table H, or comprising modulating expression in a
plant a variant of a nucleic acid encoding an orthologue, paralogue or
homologue of any of the amino acid sequences given in Table H, which
variant nucleic acid is obtained by gene shuffling.

[0644]A preferred method for modulating (preferably, increasing)
expression in a plant of such a variant obtained by gene shuffling is by
introducing and expressing in a plant a variant of any one of the nucleic
acid sequences given in Table H, or comprising introducing and expressing
in a plant a variant of a nucleic acid sequence encoding an orthologue,
paralogue or homologue of any of the amino acid sequences given in Table
H, which variant nucleic acid is obtained by gene shuffling.

[0645]Such variants obtained by gene shuffling useful in the methods of
the invention, include nucleic acids of sufficient length to encode a
polypeptide falling under the definition of a Class II HD-Zip
transcription factor, i.e. meaning that the polypeptide comprising the
following: (i) a homeodomain box; (ii) a leucine zipper; and (iii) Motifs
I, II and III described herein.

[0646]Furthermore, nucleic acids useful in the methods of the invention
may also be obtained by site-directed mutagenesis. Several methods are
available to achieve site-directed mutagenesis, the most common being
PCR-based methods (Current Protocols in Molecular Biology. Wiley Eds.).

[0647]Class II HD-Zip transcription factors exhibit the general biological
activity of transcription factors (at least in their native form) and
typically have DNA-binding activity and an activation domain. A person
skilled in the art may easily determine the presence of an activation
domain and DNA-binding activity using routine tools and techniques. Sessa
et al., 1997 (J Mol Biol 274(3):303-309) studied the DNA-binding
properties of the ATHB-1 and ATHB-2 (=HAT4) HD-Zip (HD-Zip-1 and -2)
domains and found that they interact with DNA as homodimers and recognize
two distinct 9 by pseudopalindromic sequences, CAAT(A/T)ATTG (BS-1) and
CAAT(G/C)ATTG (BS-2), respectively. From a mutational analysis of the
HD-Zip-2 domain, they determined that conserved amino acid residues of
helix 3, Va147 and Asn51, and Arg55 are essential for the DNA-binding
activity of the HD-Zip-2 domain. They also report that the preferential
recognition of a G/C base-pair at the central position by the HD-Zip-2
domain is abolished either by the replacement of Arg55 with lysine or by
the substitution of Glu46 and Thr56 with the corresponding residues of
the HD-Zip-1 domain (alanine and tryptophan, respectively).

[0648]According to a preferred feature of the invention, the modulated
expression is increased expression. Methods for increasing expression of
nucleic acids or genes, or gene products, are well documented in the art.

[0649]Another method for modulating expression in a plant of a nucleic
acid encoding a Class II HD-Zip transcription factor comprises the
reduction or substantial elimination of expression in a plant of an
endogenous gene encoding a Class II HD-Zip transcription factor.
Reference herein to "reduction or substantial elimination" is taken to
mean a decrease in endogenous gene expression and/or polypeptide levels
and/or polypeptide activity relative to control plants.

[0650]The reduction or substantial elimination is in increasing order of
preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%,
or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control
plants. Methods for decreasing expression are described above in the
"definitions" sections.

[0651]For the reduction or substantial elimination of expression an
endogenous gene in a plant, a sufficient length of substantially
contiguous nucleotides of a nucleic acid sequence is required. In order
to perform gene silencing, this may be as little as 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as
much as the entire gene (including the 5' and/or 3' UTR, either in part
or in whole). The stretch of substantially contiguous nucleotides may be
derived from SEQ ID NO: 229, or from any of the nucleic acid sequences
given in Table H, or from any nucleic acid capable of encoding an
orthologue, paralogue or homologue of any one of the amino acid sequences
given in Table H. A nucleic acid sequence encoding a (functional)
polypeptide is not a requirement for the various methods discussed herein
for the reduction or substantial elimination of expression of an
endogenous gene.

[0652]Nucleic acids suitable for use in the methods of the invention may
be derived from any natural or artificial source. The nucleic acid may be
modified from its native form in composition and/or genomic environment
through deliberate human manipulation. Preferably the nucleic acid is
from a plant, further preferably from a dicotyledonous plant, further
preferably from the family Brassicaceae, more preferably from Arabidopsis
thaliana.

[0653]A preferred method for modulating (preferably, increasing)
expression of a nucleic acid encoding a Class II HD-Zip transcription
factor is by introducing and expressing in a plant a nucleic acid
encoding a Class II HD-Zip transcription factor; however the effects of
performing the method, i.e. the modified content of seed storage
compounds, may also be achieved using other well known techniques. One
such technique is T-DNA activation tagging. The effects of the invention
may also be reproduced using the technique of TILLING. The effects of the
invention may also be reproduced using homologous recombination, which
allows introduction in a genome of a selected nucleic acid at a defined
selected position.

[0654]Performance of the methods of the invention gives plants having
seeds with a modified content of seed storage compounds relative to the
seeds of control plants. The modified content of seed storage compounds
refers to a modified content of one or more of lipids, oil, fatty acids,
starch, sugar and proteins relative to that of control plants.
Preferably, modulation of expression in a plant of a nucleic acid
encoding a Class II HD-Zip transcription factor gives plants with seeds
having increased oil content relative to the seeds of control plants. In
such a case, the modulated expression is typically overexpression in a
plant of a nucleic acid encoding a Class II HD-Zip transcription factor.
Preferably, modulation of expression in a plant of a nucleic acid
encoding a Class II HD-Zip transcription factor gives plants with seeds
having increased protein content relative to the seeds of control plants.
In such a case, the modulated expression is typically the reduction or
substantial elimination of expression of an endogenous Class II HD-Zip
transcription factor-encoding gene.

[0655]The present invention also encompasses plants or parts thereof
(particularly seeds) obtainable by the methods according to the present
invention. The plants or parts thereof comprise a nucleic acid transgene
(comprising any one of the nucleic acid sequences described above as
being useful in the methods of the invention).

[0656]The invention also provides genetic constructs and vectors to
facilitate introduction and/or expression in a plant of the nucleic acid
sequences described above as being useful in the methods of the
invention.

[0657]More particularly, there is provided a gene construct comprising:
[0658](i) A nucleic acid encoding a Class II HD-Zip transcription factor
as defined hereinabove; [0659](ii) One or more control sequences operably
linked to the nucleic acid of (i).

[0660]Constructs useful in the methods according to the present invention
may be constructed using recombinant DNA technology well known to persons
skilled in the art. The gene constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for expression of the gene of interest in the
transformed cells. The invention therefore provides use of a gene
construct as defined hereinabove in the methods of the invention.

[0661]Plants are transformed with a vector comprising the sequence of
interest. The skilled artisan is well aware of the genetic elements that
must be present in the vector in order to successfully transform, select
and propagate host cells containing the sequence of interest. The
sequence of interest is operably linked to one or more control sequences
(at least to a promoter). The terms "regulatory element", "control
sequence" and "promoter" are all used interchangeably herein and are
defined above.

[0662]Advantageously, any type of promoter, whether natural or synthetic,
may be used to drive expression of the nucleic acid sequence. See the
"Definitions" section herein for definitions of various promoter types.

[0663]According to a preferred feature of the invention, the nucleic acid
of interest is operably linked to a constitutive promoter. A constitutive
promoter is transcriptionally active during most but not necessarily all
phases of growth and development and is substantially ubiquitously
expressed. The constitutive promoter is preferably a GOS2 promoter, more
preferably the constitutive promoter is a rice GOS2 promoter, further
preferably the constitutive promoter is represented by a nucleic acid
sequence substantially similar to SEQ ID NO: 56 or SEQ ID NO: 282, most
preferably the constitutive promoter is as represented by SEQ ID NO: 56
or SEQ ID NO: 282. Examples of other constitutive promoters which may
also be used to perform the methods of the invention are shown above.

[0664]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0665]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-oh and colE1.

[0666]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). Therefore, the genetic construct
may optionally comprise a selectable marker gene. Selectable markers are
described in more detail in the "definitions" section herein. The marker
genes may be removed or excised from the transgenic cell once they are no
longer needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section.

[0667]The invention also provides a method for the production of
transgenic plants having modified content of storage compounds in seeds
relative to control plants, comprising introduction and expression in a
plant of a nucleic acid sequence represented by SEQ ID NO: 229, or
comprising introduction and expression in a plant of a nucleic acid
sequence given in Table H, or comprising introduction and expression in a
plant of a nucleic acid sequence encoding an orthologue, paralogue or
homologue of any of the amino acid sequences given in Table H, or
comprising introduction and expression in a plant of any of the nucleic
acids defined herein as being useful in the methods of the invention.

[0668]More specifically, the present invention provides a method for the
production of transgenic plants having modified content of storage
compounds in seeds relative to control plants, which method comprises:
[0669](i) introducing and expressing in a plant, plant part or plant cell
a nucleic acid sequence represented by SEQ ID NO: 229, or comprising
introducing and expressing in a plant a nucleic acid sequence given in
Table H, or comprising introducing and expressing in a plant a nucleic
acid sequence encoding an orthologue, paralogue or homologue of any of
the amino acid sequences given in Table H; and [0670](ii) cultivating the
plant cell under conditions promoting plant growth and development; and
[0671](iii) harvest of seeds from the plant of (ii); and optionally
[0672](iv) extraction of any one or more of lipids, oils, fatty acids,
starch, sugar or protein from the seeds of (iii).

[0673]The harvested seeds may be processed to extract particular seed
storage compounds, such as lipids, oils fatty acids, starch, sugar or
protein. In particular, the seeds are used for oil extraction. The oils
may be extracted from processed or unprocessed seeds.

[0674]The nucleic acid may be introduced directly into a plant cell or
into the plant itself (including introduction into a tissue, organ or any
other part of a plant). According to a preferred feature of the present
invention, the nucleic acid is preferably introduced into a plant by
transformation.

[0675]The term "transformation" as referred to herein is as defined above.
Plant tissue capable of subsequent clonal propagation, whether by
organogenesis or embryogenesis, may be transformed with a genetic
construct of the present invention and a whole plant regenerated from
there. The particular tissue chosen will vary depending on the clonal
propagation systems available for, and best suited to, the particular
species being transformed. Exemplary tissue targets include leaf disks,
pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,
existing meristematic tissue (e.g., apical meristem, axillary buds, and
root meristems), and induced meristem tissue (e.g., cotyledon meristem
and hypocotyl meristem). The polynucleotide may be transiently or stably
introduced into a host cell and may be maintained non-integrated, for
example, as a plasmid. Alternatively, it may be integrated into the host
genome. The resulting transformed plant cell may then be used to
regenerate a transformed plant in a manner known to persons skilled in
the art.

[0676]Transformation of plant species is now a fairly routine technique.
Advantageously, any of several transformation methods may be used to
introduce the gene of interest into a suitable ancestor cell.
Transformation methods include the use of liposomes, electroporation,
chemicals that increase free DNA uptake, injection of the DNA directly
into the plant, particle gun bombardment, transformation using viruses or
pollen and microprojection. Methods may be selected from the
calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.
(1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8:
363-373); electroporation of protoplasts (Shillito R. D. et al. (1985)
Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A
et al. (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle
bombardment (Klein T M et al. (1987) Nature 327: 70) infection with
(non-integrative) viruses and the like. Transgenic rice plants are
preferably produced via Agrobacterium-mediated transformation using any
of the well known methods for rice transformation, such as described in
any of the following: published European patent application EP 1198985
A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant
Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282,
1994), which disclosures are incorporated by reference herein as if fully
set forth. In the case of corn transformation, the preferred method is as
described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996)
or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures
are incorporated by reference herein as if fully set forth.

[0677]The genetically modified plant cells can be regenerated via all
methods with which the skilled worker is familiar. Suitable methods can
be found in the abovementioned publications by S. D. Kung and R. Wu,
Potrykus or Hofgen and Willmitzer.

[0678]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant. To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0679]Following DNA transfer and regeneration, putatively transformed
plants may be evaluated, for instance using Southern analysis, for the
presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels of the
newly introduced DNA may be monitored using Northern and/or Western
analysis, or quantitative PCR, all techniques being well known to persons
having ordinary skill in the art.

[0680]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
to give homozygous second generation (or T2) transformants, and the T2
plants further propagated through classical breeding techniques.

[0681]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0682]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention.

[0683]The invention also includes host cells containing any one of the
nucleic acids described above as being useful in the methods of the
invention. Preferred host cells according to the invention are plant
cells.

[0684]The methods of the invention are advantageously applicable to any
plant. Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0686]The invention also extends to harvestable parts of a plant,
particularly seeds, but also leaves, fruits, flowers, stems, rhizomes,
tubers, roots and bulbs. The invention furthermore relates to products
derived, preferably directly derived, from a harvestable part of such a
plant, such as dry pellets or powders, oil, fat and fatty acids, starch
or proteins. A particular product of interest derived from a harvestable
part of a plant is oil.

[0687]The present invention also encompasses use of any of the nucleic
acids mentioned herein as being useful in the methods of the invention in
modifying seed storage content relative to control plants. Particularly
useful is the nucleic acid represented by SEQ ID NO: 229, and any of the
nucleic acid sequences given in Table A, and any nucleic acid sequence
encoding an orthologue, paralogue or homologue of any of the polypeptide
sequences given in Table H. The present invention also encompasses use of
a polypeptide sequence represented by SEQ ID NO: 230, and use of a
polypeptide sequences given in Table H in modifying seed storage content
relative to control plants.

[0688]These nucleic acids or the encoded polypeptides may find use in
breeding programmes in which a DNA marker is identified which may be
genetically linked to a Class II HD-Zip transcription factor-encoding
gene. The nucleic acids/genes, or the polypeptides themselves may be used
to define a molecular marker. This DNA or protein marker may then be used
in breeding programmes to select plants having modified content of seed
storage compounds.

[0689]Allelic variants may also find use in marker-assisted breeding
programmes. Such breeding programmes sometimes require introduction of
allelic variation by mutagenic treatment of the plants, using for example
EMS mutagenesis; alternatively, the programme may start with a collection
of allelic variants of so called "natural" origin caused unintentionally.
Identification of allelic variants then takes place, for example, by PCR.
This is followed by a step for selection of superior allelic variants of
the sequence in question and which give increased yield. Selection is
typically carried out by monitoring growth performance of plants
containing different allelic variants of the sequence in question. Growth
performance may be monitored in a greenhouse or in the field. Further
optional steps include crossing plants in which the superior allelic
variant was identified with another plant. This could be used, for
example, to make a combination of interesting phenotypic features.

[0690]The nucleic acids may also be used as probes for genetically and
physically mapping the genes that they are a part of, and as markers for
traits linked to those genes. Such information may be useful in plant
breeding in order to develop lines with desired phenotypes. Such use of
requires only a nucleic acid sequence of at least 15 nucleotides in
length. The nucleic acids may be used as restriction fragment length
polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and
Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of
restriction-digested plant genomic DNA may be probed with the nucleic
acids. The resulting banding patterns may then be subjected to genetic
analyses using computer programs such as MapMaker (Lander et al. (1987)
Genomics 1: 174-181) in order to construct a genetic map. In addition,
the nucleic acids may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of individuals
representing parent and progeny of a defined genetic cross. Segregation
of the DNA polymorphisms is noted and used to calculate the position of
the nucleic acid in the genetic map previously obtained using this
population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0691]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0693]In another embodiment, the nucleic acid probes may be used in direct
fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favor use of
large clones (several kb to several hundred kb; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow performance
of FISH mapping using shorter probes.

[0694]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acids. Examples
include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med
11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et
al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and
Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the
sequence of a nucleic acid is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0695]The methods according to the present invention result in plants
having modified content of seed storage compounds, as described
hereinbefore. These traits may also be combined with other economically
advantageous traits, such as yield-enhancing traits, tolerance to other
abiotic and biotic stresses, traits modifying various architectural
features and/or biochemical and/or physiological features.

Detailed Description for the SYB1 Polypeptide

[0696]Surprisingly, it has now been found that modulating expression in a
plant of a nucleic acid encoding a SYB1 polypeptide gives plants having
enhanced yield-related traits relative to control plants. This yield
increase was surprisingly observed when the plants were cultivated under
conditions without stress (non-stress conditions). The particular class
of SYB1 polypeptides suitable for enhancing yield-related traits in
plants is described in detail below.

[0697]The present invention provides a method for enhancing yield-related
traits in plants relative to control plants, comprising modulating
expression in a plant of a nucleic acid encoding a SYB1 polypeptide. The
term "modulation" means in relation to expression or gene expression, a
process in which the expression level is changed by said gene expression
in comparison to the control plant, preferably the expression level is
increased. The original, unmodulated expression may be of any kind of
expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent
translation.

[0698]Any reference hereinafter to a "protein useful in the methods of the
invention" is taken to mean a SYB1 polypeptide as defined herein. Any
reference hereinafter to a "nucleic acid useful in the methods of the
invention" is taken to mean a nucleic acid capable of encoding such a
SYB1 polypeptide. The terms "polypeptide" and "protein" are used
interchangeably herein and refer to amino acids in a polymeric form of
any length. The terms "polynucleotide(s)", "nucleic acid sequence(s)",
"nucleotide sequence(s)" are used interchangeably herein and refer to
nucleotides, either ribonucleotides or deoxyribonucleotides or a
combination of both, in a polymeric form of any length.

[0699]A preferred method for modulating (preferably, increasing)
expression of a nucleic acid encoding a protein useful in the methods of
the invention is by introducing and expressing in a plant a nucleic acid
encoding a protein useful in the methods of the invention as defined
below.

[0700]The nucleic acid to be introduced into a plant (and therefore useful
in performing the methods of the invention) is any nucleic acid encoding
the type of protein which will now be described, hereafter also named
"SYB1 nucleic acid" or "SYB1 gene". A "SYB1" polypeptide as defined
herein refers to any amino acid sequence comprising 3 Zinc finger domains
of the RanBP type (SMART accession number: SM00547, Interpro accession
number: IPR001876) and optionally one or more low complexity domain(s),
but lacking, when analysed against the SMART database, any other domains
that do not overlap with the Zinc finger domains or, if present, the low
complexity domain. The ZnF_RBZ type Zinc finger domain is present in
Ran-binding proteins (RanBPs), and other proteins. In RanBPs, this domain
binds RanGDP. Low complexity domains may be identified using the SEG
algorithm of Wootton and Federhen (Methods Enzymol. 266 (1996), pp.
554-571). Preferably SYB1 polypeptides in their natural form (that is, as
they occur in nature) are in increasing order of preference, no longer
then 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 190 or 180
amino acids, more preferably the length of SYB1 polypeptide ranges
between 180 and 130 amino acids.

[0701]Zinc finger domains are known to bind zinc ions and are generally
involved in protein-DNA or protein-protein interactions. Preferably, the
Zinc finger domains in the SYB1 protein start with one of the following
motifs: (G/R/D/N)DW (motif 1, SEQ ID NO: 344), or GSW (motif 2, SEQ ID
NO: 345), and has a first conserved cysteine residue on position 5
(wherein positions 1 to 3 are taken by motif 1 or motif 2), a second
conserved cysteine residue between positions 7 and 10, a third conserved
cysteine residue between positions 18 and 21 and a fourth conserved
cysteine residue between positions 21 and 24. Furthermore, the Zinc
finger domain in the SYB1 protein preferably comprises the conserved
motifs NF(Q/C/S)(R/K)R (motif 3, SEQ ID NO: 346) or N(F/Y)(A/S/P)(N/S/F)R
(motif 4, SEQ ID NO 347).

[0702]Optionally, the sequence located between the Zn-finger domains (say
starting after the fourth conserved cysteine residue and ending before
the start of motif 1 or 2) is enriched in glycine residues, the glycine
content may be as high as 35%, or even higher (whereas the glycine
content in an average protein is around 6.93% (SWISS PROT notes, release
44, July 2004)). Additionally or alternatively, the serine content in
this sequence may also be increased compared to the serine content in an
average protein (6.89%).

[0703]Preferably, the polypeptide sequence which when used in the
construction of a phylogenetic tree, such as the one depicted in FIG. 2b,
clusters with the group of SYB1 polypeptides comprising the amino acid
sequence represented by SEQ ID NO: 286 rather than with any other group.

[0704]Examples of polypeptides useful in the methods of the invention and
nucleic acids encoding the same are as given below in the table of
Example 40.

[0705]Also useful in the methods of the invention are homologues of any
one of the amino acid sequences given in the table of Example 40, and
derivatives of any one of the polypeptides given in the table of Example
40 or orthologues or paralogues of any of the aforementioned SEQ ID NOs.

[0706]The invention is illustrated by transforming plants with the
Arabidopsis thaliana nucleic acid sequence represented by SEQ ID NO: 285,
encoding the polypeptide sequence of SEQ ID NO: 286, however performance
of the invention is not restricted to these sequences. The methods of the
invention may advantageously be performed using any nucleic acid encoding
a protein useful in the methods of the invention as defined herein,
including orthologues and paralogues, such as any of the nucleic acid
sequences given in the table of Example 40. The amino acid sequences
given in the table of Example 40 may be considered to be orthologues and
paralogues of the SYB1 polypeptide represented by SEQ ID NO: 286.

[0707]Orthologues and paralogues may easily be found by performing a
so-called reciprocal blast search. Typically, this involves a first BLAST
involving BLASTing a query sequence (for example using any of the
sequences listed in the table of Example 40) against any sequence
database, such as the publicly available NCBI database. BLASTN or TBLASTX
(using standard default values) are generally used when starting from a
nucleotide sequence, and BLASTP or TBLASTN (using standard default
values) when starting from a protein sequence. The BLAST results may
optionally be filtered. The full-length sequences of either the filtered
results or non-filtered results are then BLASTed back (second BLAST)
against sequences from the organism from which the query sequence is
derived (where the query sequence is SEQ ID NO: 285 or SEQ ID NO: 286,
the second BLAST would therefore be against Arabidopsis thaliana
sequences). The results of the first and second BLASTs are then compared.
A paralogue is identified if a high-ranking hit from the first blast is
from the same species as from which the query sequence is derived, a
BLAST back then ideally results in the query sequence as highest hit; an
orthologue is identified if a high-ranking hit in the first BLAST is not
from the same species as from which the query sequence is derived, and
preferably results upon BLAST back in the query sequence being among the
highest hits.

[0708]High-ranking hits are those having a low E-value. The lower the
E-value, the more significant the score (or in other words the lower the
chance that the hit was found by chance). Computation of the E-value is
well known in the art. In addition to E-values, comparisons are also
scored by percentage identity. Percentage identity refers to the number
of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length. In the
case of large families, ClustalW may be used, followed by a neighbour
joining tree, to help visualize clustering of related genes and to
identify orthologues and paralogues.

[0709]The table of Example 40 gives examples of orthologues and paralogues
of the SYB1 protein represented by SEQ ID NO 286. Further orthologues and
paralogues may readily be identified using the BLAST procedure described
above.

[0711]Domains may also be identified using routine techniques, such as by
sequence alignment. Methods for the alignment of sequences for comparison
are well known in the art, such methods include GAP, BESTFIT, BLAST,
FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970)
J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete
sequences) alignment of two sequences that maximizes the number of
matches and minimizes the number of gaps. The BLAST algorithm (Altschul
et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence
identity and performs a statistical analysis of the similarity between
the two sequences. The software for performing BLAST analysis is publicly
available through the National Centre for Biotechnology Information
(NCBI). Homologues may readily be identified using, for example, the
ClustalW multiple sequence alignment algorithm (version 1.83), with the
default pairwise alignment parameters, and a scoring method in
percentage. Global percentages of similarity and identity may also be
determined using one of the methods available in the MatGAT software
package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29.
MatGAT: an application that generates similarity/identity matrices using
protein or DNA sequences.). Minor manual editing may be performed to
optimise alignment between conserved motifs, as would be apparent to a
person skilled in the art. Furthermore, instead of using full-length
sequences for the identification of homologues, specific domains (such as
the ZnF_RBZ type Zinc finger domain, or one of the motifs defined above)
may be used as well. The sequence identity values, which are indicated
below in Example 42 as a percentage were determined over the entire
nucleic acid or amino acid sequence, and/or over selected domains or
conserved motif(s), using the programs mentioned above using the default
parameters. Furthermore, SYB1 proteins (at least in their native form)
may interact with proteins or nucleic acids and has the effect of
increasing seed yield when expressed according to the methods of the
present invention. Further details are provided in Example 45.

[0712]Nucleic acids encoding proteins useful in the methods of the
invention need not be full-length nucleic acids, since performance of the
methods of the invention does not rely on the use of full-length nucleic
acid sequences. Examples of nucleic acids suitable for use in performing
the methods of the invention include the nucleic acid sequences given in
the table of Example 40, but are not limited to those sequences. Nucleic
acid variants may also be useful in practising the methods of the
invention. Examples of such nucleic acid variants include portions of
nucleic acids encoding a protein useful in the methods of the invention,
nucleic acids hybridising to nucleic acids encoding a protein useful in
the methods of the invention, splice variants of nucleic acids encoding a
protein useful in the methods of the invention, allelic variants of
nucleic acids encoding a protein useful in the methods of the invention
and variants of nucleic acids encoding a protein useful in the methods of
the invention that are obtained by gene shuffling. The terms portion,
hybridising sequence, splice variant, allelic variant and gene shuffling
will now be described.

[0713]According to the present invention, there is provided a method for
enhancing yield-related traits in plants, comprising introducing and
expressing in a plant a portion of any one of the nucleic acid sequences
given in the table of Example 40, or a portion of a nucleic acid encoding
an orthologue, paralogue or homologue of any of the amino acid sequences
given in the table of Example 40.

[0714]Portions useful in the methods of the invention, encode a
polypeptide falling within the definition of a nucleic acid encoding a
protein useful in the methods of the invention as defined herein and
having substantially the same biological activity as the amino acid
sequences given in the table of Example 40. Preferably, the portion is a
portion of any one of the nucleic acids given in the table of Example 40.
The portion is typically at least 200 consecutive nucleotides in length,
preferably at least 300 consecutive nucleotides in length, more
preferably at least 400 consecutive nucleotides in length and most
preferably at least 500 consecutive nucleotides in length, the
consecutive nucleotides being of any one of the nucleic acid sequences
given in the table of Example 40. Most preferably the portion is a
portion of the nucleic acid of SEQ ID NO: 285. Preferably, the portion
encodes an amino acid sequence comprising three ZnF_RBZ type Zinc finger
domains as defined herein. Preferably, the portion encodes an amino acid
sequence which when used in the construction of a SYB1 phylogenetic tree,
such as the one depicted in FIG. 23b, tends to cluster with the group of
SYB1 proteins comprising the amino acid sequence represented by SEQ ID
NO: 286 rather than with any other group.

[0715]A portion of a nucleic acid encoding a SYB1 protein as defined
herein may be prepared, for example, by making one or more deletions to
the nucleic acid. The portions may be used in isolated form or they may
be fused to other coding (or non coding) sequences in order to, for
example, produce a protein that combines several activities. When fused
to other coding sequences, the resultant polypeptide produced upon
translation may be bigger than that predicted for the SYB1 protein
portion.

[0716]Another nucleic acid variant useful in the methods of the invention
is a nucleic acid capable of hybridising, under reduced stringency
conditions, preferably under stringent conditions, with a nucleic acid
encoding a SYB1 protein as defined herein, or with a portion as defined
herein.

[0717]Hybridising sequences useful in the methods of the invention, encode
a polypeptide having three ZnF_RBZ type Zinc finger domains (see the
alignment of FIG. 23a) and having substantially the same biological
activity as the SYB1 protein represented by any of the amino acid
sequences given in the table of Example 40. The hybridising sequence is
typically at least 200 consecutive nucleotides in length, preferably at
least 300 consecutive nucleotides in length, more preferably at least 400
consecutive nucleotides in length and most preferably at least 500
consecutive nucleotides in length, the consecutive nucleotides being of
any one of the nucleic acid sequences given in the table of Example 40.
Preferably, the hybridising sequence is one that is capable of
hybridising to any of the nucleic acids given in the table of Example 40,
or to a portion of any of these sequences, a portion being as defined
above. Most preferably, the hybridising sequence is capable of
hybridising to a nucleic acid as represented by SEQ ID NO: 285 or to a
portion thereof. Preferably, the hybridising sequence encodes an amino
acid sequence comprising any one or more of the motifs or domains as
defined herein. Preferably, the hybridising sequence encodes an amino
acid sequence which when used in the construction of a SYB1 phylogenetic
tree, such as the one depicted in FIG. 23b, tends to cluster with the
group of SYB1 proteins comprising the amino acid sequence represented by
SEQ ID NO: 286 rather than with any other group.

[0718]According to the present invention, there is provided a method for
enhancing yield-related traits in plants, comprising introducing and
expressing in a plant a nucleic acid capable of hybridizing to any one of
the nucleic acids given in the table of Example 40, or comprising
introducing and expressing in a plant a nucleic acid capable of
hybridising to a nucleic acid encoding an orthologue, paralogue or
homologue of any of the nucleic acid sequences given in the table of
Example 40.

[0719]Another nucleic acid variant useful in the methods of the invention
is a splice variant encoding a SYB1 protein as defined hereinabove, the
term "splice variant" being as defined above.

[0720]According to the present invention, there is provided a method for
enhancing yield-related traits in plants, comprising introducing and
expressing in a plant a splice variant of any one of the nucleic acid
sequences given in the table of Example 40, or a splice variant of a
nucleic acid encoding an orthologue, paralogue or homologue of any of the
amino acid sequences given in the table of Example 40.

[0721]Preferred splice variants are splice variants of a nucleic acid
represented by SEQ ID NO: 285 or a splice variant of a nucleic acid
encoding an orthologue or paralogue of SEQ ID NO: 286. Preferably, the
amino acid sequence encoded by the splice variant comprises any one or
more of the motifs or domains as defined herein. Preferably, the amino
acid sequence encoded by the splice variant, when used in the
construction of a SYB1 phylogenetic tree, such as the one depicted in
FIG. 23b, tends to cluster with the group of SYB1 proteins comprising the
amino acid sequence represented by SEQ ID NO: 286 rather than with any
other group.

[0722]Another nucleic acid variant useful in performing the methods of the
invention is an allelic variant of a nucleic acid encoding a SYB1 protein
as defined hereinabove. Allelic variants exist in nature, and encompassed
within the methods of the present invention is the use of these natural
alleles. The allelic variants useful in the methods of the present
invention have substantially the same biological activity as the SYB1
protein of SEQ ID NO: 286.

[0723]According to the present invention, there is provided a method for
enhancing yield-related traits in plants, comprising introducing and
expressing in a plant an allelic variant of any one of the nucleic acids
given in the table of Example 40, or comprising introducing and
expressing in a plant an allelic variant of a nucleic acid encoding an
orthologue, paralogue or homologue of any of the amino acid sequences
given in the table of Example 40.

[0724]Preferably, the allelic variant is an allelic variant of SEQ ID NO:
285 or an allelic variant of a nucleic acid encoding an orthologue or
paralogue of SEQ ID NO: 286. Preferably, the amino acid sequence encoded
by the allelic variant comprises any one or more of the motifs or domains
as defined herein. Preferably, the amino acid sequence encoded by the
allelic variant, when used in the construction of a SYB1 phylogenetic
tree, such as the one depicted in FIG. 23b, tends to cluster with the
group of SYB1 proteins comprising the amino acid sequence represented by
SEQ ID NO: 286 rather than with any other group.

[0725]A further nucleic acid variant useful in the methods of the
invention is a nucleic acid variant obtained by gene shuffling, the term
"gene shuffling" as described above.

[0726]According to the present invention, there is provided a method for
enhancing yield-related traits in plants, comprising introducing and
expressing in a plant a variant of any one of the nucleic acid sequences
given in the table of Example 40, or comprising introducing and
expressing in a plant a variant of a nucleic acid encoding an orthologue,
paralogue or homologue of any of the amino acid sequences given in the
table of Example 40, which variant nucleic acid is obtained by gene
shuffling.

[0727]Preferably, the variant nucleic acid obtained by gene shuffling
encodes an amino acid sequence comprising any one or more of the motifs
or domains as defined herein. Preferably, the amino acid sequence encoded
by the variant nucleic acid obtained by gene shuffling, when used in the
construction of a SYB1 phylogenetic tree such as the one depicted in FIG.
23b, tends to cluster with the group of SYB1 proteins comprising the
amino acid sequence represented by SEQ ID NO: 286 rather than with any
other group.

[0728]Furthermore, nucleic acid variants may also be obtained by
site-directed mutagenesis. Several methods are available to achieve
site-directed mutagenesis, the most common being PCR based methods
(Current Protocols in Molecular Biology. Wiley Eds.).

[0729]Nucleic acids encoding SYB1 proteins may be derived from any natural
or artificial source. The nucleic acid may be modified from its native
form in composition and/or genomic environment through deliberate human
manipulation. Preferably the SYB1-encoding nucleic acid is from a plant,
further preferably from a dicotyledonous plant, more preferably from the
Brassicaceae family; most preferably the nucleic acid is from Arabidopsis
thaliana.

[0730]Any reference herein to a SYB1 protein is therefore taken to mean a
SYB1 protein as defined above. Any nucleic acid encoding such a SYB1
protein is suitable for use in performing the methods of the invention.

[0731]The present invention also encompasses plants or parts thereof
(including seeds) obtainable by the methods according to the present
invention. The plants or parts thereof comprise a nucleic acid transgene
encoding a SYB1 protein as defined above.

[0732]The invention also provides genetic constructs and vectors to
facilitate introduction and/or expression of the nucleic acid sequences
useful in the methods according to the invention, in a plant. Constructs
useful in the methods according to the present invention may be
constructed using recombinant DNA technology well known to persons
skilled in the art. The gene constructs may be inserted into vectors,
which may be commercially available, suitable for transforming into
plants and suitable for expression of the gene of interest in the
transformed cells. The invention also provides use of a gene construct as
defined herein in the methods of the invention.

[0733]More specifically, the present invention provides a construct
comprising: [0734](i) a SYB1 nucleic acid or variant thereof, as
defined hereinabove; [0735](ii) one or more control sequences operably
linked the nucleic acid sequence of (i); and optionally [0736](iii) a
transcription termination sequence.

[0737]Preferably, the nucleic acid encoding a SYB1 polypeptide is as
defined above. The term "control sequence" and "termination sequence" are
as defined herein.

[0738]Plants are transformed with a vector comprising the sequence of
interest (i.e., a nucleic acid encoding a SYB1 polypeptide as defined
herein. The skilled artisan is well aware of the genetic elements that
must be present on the vector in order to successfully transform, select
and propagate host cells containing the sequence of interest. The
sequence of interest is operably linked to one or more control sequences
(at least to a promoter). The terms "regulatory element", "control
sequence" and "promoter" are all used interchangeably herein and are
defined above.

[0739]Advantageously, any type of promoter may be used to drive expression
of the nucleic acid sequence. Preferably, the SYB1 nucleic acid or
variant thereof is operably linked to a constitutive promoter. A
preferred constitutive promoter is one that is also substantially
ubiquitously expressed. Further preferably the promoter is derived from a
plant, more preferably a monocotyledonous plant. Most preferred is use of
a GOS2 promoter (from rice) (SEQ ID NO: 56 or 343). It should be clear
that the applicability of the present invention is not restricted to the
SYB1 nucleic acid represented by SEQ ID NO: 285, nor is the applicability
of the invention restricted to expression of a SYB1 nucleic acid when
driven by a GOS2 promoter. Examples of other constitutive promoters which
may also be used to drive expression of a SYB1 nucleic acid are shown
above.

[0740]Optionally, one or more terminator sequences may be used in the
construct introduced into a plant. Additional regulatory elements may
include transcriptional as well as translational enhancers. Those skilled
in the art will be aware of terminator and enhancer sequences that may be
suitable for use in performing the invention. An intron sequence may also
be added to the 5' untranslated region (UTR) or in the coding sequence to
increase the amount of the mature message that accumulates in the
cytosol, as described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or
5'UTR regions) may be protein and/or RNA stabilizing elements. Such
sequences would be known or may readily be obtained by a person skilled
in the art.

[0741]The genetic constructs of the invention may further include an
origin of replication sequence that is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an episomal
genetic element (e.g. plasmid or cosmid molecule). Preferred origins of
replication include, but are not limited to, the f1-ori and colE1.

[0742]For the detection of the successful transfer of the nucleic acid
sequences as used in the methods of the invention and/or selection of
transgenic plants comprising these nucleic acids, it is advantageous to
use marker genes (or reporter genes). "Selectable markers" are described
in more detail in the definitions section. The marker genes may be
removed or excised from the transgenic cell once they are no longer
needed. Techniques for marker removal are known in the art, useful
techniques are described above in the definitions section.

[0743]The invention also provides a method for the production of
transgenic plants having enhanced yield-related traits relative to
control plants, comprising introduction and expression in a plant of any
nucleic acid encoding a SYB1 protein as defined hereinabove.

[0744]More specifically, the present invention provides a method for the
production of transgenic plants having increased yield, which method
comprises: [0745](i) introducing and expressing in a plant or plant
cell a SYB1 nucleic acid or variant thereof; and [0746](ii) cultivating
the plant cell under conditions promoting plant growth and development.

[0747]The nucleic acid of (i) may be any of the nucleic acids capable of
encoding a SYB1 polypeptide as defined herein.

[0748]The nucleic acid may be introduced directly into a plant cell or
into the plant itself (including introduction into a tissue, organ or any
other part of a plant). According to a preferred feature of the present
invention, the nucleic acid is preferably introduced into a plant by
transformation. The term "transformation" is described in more detail in
the "definitions" section herein.

[0749]The genetically modified plant cells can be regenerated via all
methods with which the skilled worker is familiar. Suitable methods can
be found in the abovementioned publications by S.D. Kung and R. Wu,
Potrykus or Hofgen and Willmitzer.

[0750]Generally after transformation, plant cells or cell groupings are
selected for the presence of one or more markers which are encoded by
plant-expressible genes co-transferred with the gene of interest,
following which the transformed material is regenerated into a whole
plant. To select transformed plants, the plant material obtained in the
transformation is, as a rule, subjected to selective conditions so that
transformed plants can be distinguished from untransformed plants. For
example, the seeds obtained in the above-described manner can be planted
and, after an initial growing period, subjected to a suitable selection
by spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into plants.
Alternatively, the transformed plants are screened for the presence of a
selectable marker such as the ones described above.

[0751]Following DNA transfer and regeneration, putatively transformed
plants may also be evaluated, for instance using Southern analysis, for
the presence of the gene of interest, copy number and/or genomic
organisation. Alternatively or additionally, expression levels of the
newly introduced DNA may be monitored using Northern and/or Western
analysis, both techniques being well known to persons having ordinary
skill in the art.

[0752]The generated transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed
and homozygous second-generation (or T2) transformants selected, and the
T2 plants may then further be propagated through classical breeding
techniques.

[0753]The generated transformed organisms may take a variety of forms. For
example, they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootstock grafted to an untransformed
scion).

[0754]The present invention clearly extends to any plant cell or plant
produced by any of the methods described herein, and to all plant parts
and propagules thereof. The present invention extends further to
encompass the progeny of a primary transformed or transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned methods, the only requirement being that progeny exhibit
the same genotypic and/or phenotypic characteristic(s) as those produced
by the parent in the methods according to the invention.

[0755]The invention also includes host cells containing an isolated
nucleic acid encoding a SYB1 protein as defined hereinabove. Preferred
host cells according to the invention are plant cells.

[0756]Host plants for the nucleic acids or the vector used in the method
according to the invention, the expression cassette or construct or
vector are, in principle, advantageously all plants, which are capable of
synthesizing the polypeptides used in the inventive method.

[0757]The methods of the invention are advantageously applicable to any
plant. Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous plants
including fodder or forage legumes, ornamental plants, food crops, trees
or shrubs. According to a preferred embodiment of the present invention,
the plant is a crop plant. Examples of crop plants include soybean,
sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco.
Further preferably, the plant is a monocotyledonous plant. Examples of
monocotyledonous plants include sugarcane. More preferably the plant is a
cereal. Examples of cereals include rice, maize, wheat, barley, millet,
rye, triticale, sorghum and oats.

[0758]The invention also extends to harvestable parts of a plant such as,
but not limited to seeds, leaves, fruits, flowers, stems, rhizomes,
tubers and bulbs. The invention furthermore relates to products derived,
preferably directly derived, from a harvestable part of such a plant,
such as dry pellets or powders, oil, fat and fatty acids, starch or
proteins.

[0759]According to a preferred feature of the invention, the modulated
expression is increased expression. As mentioned above, a preferred
method for modulating (preferably, increasing) expression of a nucleic
acid encoding a SYB1 protein is by introducing and expressing in a plant
a nucleic acid encoding a SYB1 protein; however the effects of performing
the method, i.e. enhancing yield-related traits may also be achieved
using other well known techniques. One such technique is T-DNA activation
tagging. The effects of the invention may also be reproduced using the
technique of TILLING, or with homologous recombination, which allows
introduction in a genome of a selected nucleic acid at a defined selected
position.

[0760]Performance of the methods of the invention gives plants having
enhanced yield-related traits. In particular performance of the methods
of the invention gives plants having increased yield, especially
increased seed yield relative to control plants. The terms "yield" and
"seed yield" are described in more detail in the "definitions" section
herein.

[0761]Reference herein to enhanced yield-related traits is taken to mean
an increase in biomass (weight) of one or more parts of a plant, which
may include aboveground (harvestable) parts and/or (harvestable) parts
below ground.

[0762]In particular, such harvestable parts are seeds, and performance of
the methods of the invention results in plants having increased seed
yield relative to the seed yield of suitable control plants.

[0763]Taking corn as an example, a yield increase may be manifested as one
or more of the following: increase in the number of plants established
per square meter, an increase in the number of ears per plant, an
increase in the number of rows, number of kernels per row, kernel weight,
thousand kernel weight, ear length/diameter, increase in the seed filling
rate (which is the number of filled seeds divided by the total number of
seeds and multiplied by 100), among others. Taking rice as an example, a
yield increase may manifest itself as an increase in one or more of the
following: number of plants per square meter, number of panicles per
plant, number of spikelets per panicle, number of flowers (florets) per
panicle (which is expressed as a ratio of the number of filled seeds over
the number of primary panicles), increase in the seed filling rate (which
is the number of filled seeds divided by the total number of seeds and
multiplied by 100), increase in thousand kernel weight, among others.

[0764]Since the transgenic plants according to the present invention have
increased yield, it is likely that these plants exhibit an increased
growth rate (during at least part of their life cycle), relative to the
growth rate of control plants at a corresponding stage in their life
cycle. The increased growth rate may be specific to one or more parts of
a plant (including seeds), or may be throughout substantially the whole
plant. Plants having an increased growth rate may have a shorter life
cycle. The life cycle of a plant may be taken to mean the time needed to
grow from a dry mature seed up to the stage where the plant has produced
dry mature seeds, similar to the starting material. This life cycle may
be influenced by factors such as early vigour, growth rate, greenness
index, flowering time and speed of seed maturation. The increase in
growth rate may take place at one or more stages in the life cycle of a
plant or during substantially the whole plant life cycle. Increased
growth rate during the early stages in the life cycle of a plant may
reflect enhanced vigour. The increase in growth rate may alter the
harvest cycle of a plant allowing plants to be sown later and/or
harvested sooner than would otherwise be possible (a similar effect may
be obtained with earlier flowering time). If the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
the same plant species (for example sowing and harvesting of rice plants
followed by sowing and harvesting of further rice plants all within one
conventional growing period). Similarly, if the growth rate is
sufficiently increased, it may allow for the further sowing of seeds of
different plants species (for example the planting and harvesting of corn
plants followed by, for example, the planting and optional harvesting of
soy bean, potato or any other suitable plant). Harvesting additional
times from the same rootstock in the case of some crop plants may also be
possible. Altering the harvest cycle of a plant may lead to an increase
in annual biomass production per square meter (due to an increase in the
number of times (say in a year) that any particular plant may be grown
and harvested). An increase in growth rate may also allow for the
cultivation of transgenic plants in a wider geographical area than their
wild-type counterparts, since the territorial limitations for growing a
crop are often determined by adverse environmental conditions either at
the time of planting (early season) or at the time of harvesting (late
season). Such adverse conditions may be avoided if the harvest cycle is
shortened. The growth rate may be determined by deriving various
parameters from growth curves, such parameters may be: T-Mid (the time
taken for plants to reach 50% of their maximal size) and T-90 (time taken
for plants to reach 90% of their maximal size), amongst others.

[0765]According to a preferred feature of the present invention,
performance of the methods of the invention gives plants having an
increased growth rate relative to control plants. Therefore, according to
the present invention, there is provided a method for increasing the
growth rate of plants, which method comprises modulating expression,
preferably increasing expression, in a plant of a nucleic acid encoding a
SYB1 protein as defined herein.

[0766]An increase in yield and/or growth rate occurs whether the plant is
under non-stress conditions or whether the plant is exposed to various
stresses compared to control plants. Plants typically respond to exposure
to stress by growing more slowly. In conditions of severe stress, the
plant may even stop growing altogether. Mild stress on the other hand is
defined herein as being any stress to which a plant is exposed which does
not result in the plant ceasing to grow altogether without the capacity
to resume growth. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%, 35% or
30%, preferably less than 25%, 20% or 15%, more preferably less than 14%,
13%, 12%, 11% or 10% or less in comparison to the control plant under
non-stress conditions. Due to advances in agricultural practices
(irrigation, fertilization, pesticide treatments) severe stresses are not
often encountered in cultivated crop plants. As a consequence, the
compromised growth induced by mild stress is often an undesirable feature
for agriculture. Mild stresses are the everyday biotic and/or abiotic
(environmental) stresses to which a plant is exposed. Abiotic stresses
may be due to drought or excess water, anaerobic stress, salt stress,
chemical toxicity, oxidative stress and hot, cold or freezing
temperatures. The abiotic stress may be an osmotic stress caused by a
water stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses caused
by pathogens, such as bacteria, viruses, fungi and insects.

[0767]In particular, the methods of the present invention may be performed
under non-stress conditions or under conditions of mild drought to give
plants having increased yield relative to control plants. As reported in
Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series
of morphological, physiological, biochemical and molecular changes that
adversely affect plant growth and productivity. Drought, salinity,
extreme temperatures and oxidative stress are known to be interconnected
and may induce growth and cellular damage through similar mechanisms.
Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a
particularly high degree of "cross talk" between drought stress and
high-salinity stress. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption of
homeostasis and ion distribution in the cell. Oxidative stress, which
frequently accompanies high or low temperature, salinity or drought
stress, may cause denaturing of functional and structural proteins. As a
consequence, these diverse environmental stresses often activate similar
cell signaling pathways and cellular responses, such as the production of
stress proteins, up-regulation of anti-oxidants, accumulation of
compatible solutes and growth arrest. The term "non-stress" conditions as
used herein are those environmental conditions that allow optimal growth
of plants. Persons skilled in the art are aware of normal soil conditions
and climatic conditions for a given location.

[0768]Performance of the methods of the invention gives plants grown under
non-stress conditions or under mild drought conditions increased yield
relative to suitable control plants grown under comparable conditions.
Therefore, according to the present invention, there is provided a method
for increasing yield in plants grown under non-stress conditions or under
mild drought conditions, which method comprises increasing expression in
a plant of a nucleic acid encoding a SYB1 polypeptide.

[0769]Performance of the methods of the invention gives plants grown under
conditions of nutrient deficiency, particularly under conditions of
nitrogen deficiency, increased yield relative to control plants grown
under comparable conditions. Therefore, according to the present
invention, there is provided a method for increasing yield in plants
grown under conditions of nutrient deficiency, which method comprises
increasing expression in a plant of a nucleic acid encoding a SYB1
polypeptide. Nutrient deficiency may result from a lack of nutrients such
as nitrogen, phosphates and other phosphorous-containing compounds,
potassium, calcium, cadmium, magnesium, manganese, iron and boron,
amongst others.

[0770]In a preferred embodiment of the invention, the increase in yield
and/or growth rate occurs according to the methods of the present
invention under non-stress conditions.

[0771]The present invention also encompasses use of nucleic acids encoding
the SYB1 protein described herein and use of these SYB1 proteins in
enhancing yield-related traits in plants. The present invention
furthermore encompasses use of plants

[0772]Nucleic acids encoding the SYB1 protein described herein, or the
SYB1 proteins themselves, may find use in breeding programmes in which a
DNA marker is identified which may be genetically linked to a
SYB1-encoding gene. The nucleic acids/genes, or the SYB1 proteins
themselves may be used to define a molecular marker. This DNA or protein
marker may then be used in breeding programmes to select plants having
enhanced yield-related traits as defined hereinabove in the methods of
the invention.

[0773]Allelic variants of a SYB1 protein-encoding nucleic acid/gene may
also find use in marker-assisted breeding programmes. Such breeding
programmes sometimes require introduction of allelic variation by
mutagenic treatment of the plants, using for example EMS mutagenesis;
alternatively, the programme may start with a collection of allelic
variants of so called "natural" origin caused unintentionally.
Identification of allelic variants then takes place, for example, by PCR.
This is followed by a step for selection of superior allelic variants of
the sequence in question and which give increased yield. Selection is
typically carried out by monitoring growth performance of plants
containing different allelic variants of the sequence in question. Growth
performance may be monitored in a greenhouse or in the field. Further
optional steps include crossing plants in which the superior allelic
variant was identified with another plant. This could be used, for
example, to make a combination of interesting phenotypic features.

[0774]Nucleic acids encoding SYB1 proteins may also be used as probes for
genetically and physically mapping the genes that they are a part of, and
as markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. Such use of SYB1 protein-encoding nucleic acids requires only
a nucleic acid sequence of at least 15 nucleotides in length. The SYB1
protein-encoding nucleic acids may be used as restriction fragment length
polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch EF and
Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of
restriction-digested plant genomic DNA may be probed with the SYB1
protein-encoding nucleic acids. The resulting banding patterns may then
be subjected to genetic analyses using computer programs such as MapMaker
(Lander et al. (1987) Genomics 1: 174-181) in order to construct a
genetic map. In addition, the nucleic acids may be used to probe Southern
blots containing restriction endonuclease-treated genomic DNAs of a set
of individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the SYB1 protein-encoding nucleic acid in the
genetic map previously obtained using this population (Botstein et al.
(1980) Am. J. Hum. Genet. 32:314-331).

[0775]The production and use of plant gene-derived probes for use in
genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping
of specific cDNA clones using the methodology outlined above or
variations thereof. For example, F2 intercross populations, backcross
populations, randomly mated populations, near isogenic lines, and other
sets of individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.

[0777]In another embodiment, the nucleic acid probes may be used in direct
fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favour use of
large clones (several kb to several hundred kb; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow performance
of FISH mapping using shorter probes.

[0778]A variety of nucleic acid amplification-based methods for genetic
and physical mapping may be carried out using the nucleic acids. Examples
include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med
11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et
al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and
Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the
sequence of a nucleic acid is used to design and produce primer pairs for
use in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the mapping
cross in the region corresponding to the instant nucleic acid sequence.
This, however, is generally not necessary for mapping methods.

[0779]The methods according to the present invention result in plants
having enhanced yield-related traits, as described hereinbefore. These
traits may also be combined with other economically advantageous traits,
such as further yield-enhancing traits, tolerance to other abiotic and
biotic stresses, traits modifying various architectural features and/or
biochemical and/or physiological features.

DESCRIPTION OF FIGURES

[0780]The present invention will now be described with reference to the
following figures in which:

[0781]FIG. 1 shows an example of the domain structure of an AZ
polypeptide. The protein encoded by SEQ ID NO: 2 comprises two ankyrin
repeats (bold, underlined) and two C3H1 domains (italics, underlined).

[0782]FIG. 2 shows the Ankyrin (ANK) and C3H1 Zinc finger (C3H1) consensus
sequences according to the SMART database, the symbols for the various
amino acid groups are given in the legend.

[0784]FIG. 4 shows the binary vector p056, for expression in Oryza sativa
of an Arabidopsis thaliana AZ coding sequence under the control of a
WSI18 promoter (internal reference PRO0151).

[0785]FIG. 5 shows the typical domain structure of SYT polypeptides from
plants and mammals. The conserved SNH domain is located at the N-terminal
end of the polypeptide. The C-terminal remainder of the polypeptide
consists of a QG-rich domain in plant SYT polypeptides, and of a
QPGY-rich domain in mammalian SYT polypeptides. A Met-rich domain is
typically comprised within the first half of the QG-rich (from the N-term
to the C-term) in plants or QPGY-rich in mammals. A second Met-rich
domain may precede the SNH domain in plant SYT polypeptides

[0786]FIG. 6 shows a multiple alignment of the N-terminal end of several
SYT polypeptides, using VNTI AlignX multiple alignment program, based on
a modified ClustalW algorithm (InforMax, Bethesda, Md.,
http://www.informaxinc.com), with default settings for gap opening
penalty of 10 and a gap extension of 0.05). The SNH domain is boxed
across the plant and human SYT polypeptides. The last line in the
alignment consists of a consensus sequence derived from the aligned
sequences.

[0787]FIG. 7 shows a multiple alignment of several plant SYT polypeptides,
using VNTI AlignX multiple alignment program, based on a modified
ClustalW algorithm (InforMax, Bethesda, Md., http://www.informaxinc.com),
with default settings for gap opening penalty of 10 and a gap extension
of 0.05). The two main domains, from N-terminal to C-terminal, are boxed
and identified as SNH domain and the Met-rich/QG-rich domain.
Additionally, the N-terminal Met-rich domain is also boxed, and the
positions of SEQ ID NO: 90 and SEQ ID NO 91 are underlined in bold.

[0788]FIG. 8 shows a Neighbour joining tree resulting from the alignment
of multiple SYT polypeptides using CLUSTALW 1.83
(http://align.genome.jp/sit-bin/clustalw). The SYT1 and SYT2/SYT3 clades
are identified with brackets.

[0789]FIG. 9 shows a binary vector p0523, for expression in Oryza sativa
of an Arabidopsis thaliana AtSYT1 under the control of a GOS2 promoter
(internal reference PR00129).

[0790]FIG. 10 is an overview of the Calvin cycle. The thirteen enzymatic
reactions are shown, as well as the enzyme names that perform these
reactions. The black arrow shows the position of cpFBPase in the cycle.

[0791]FIG. 11 is a scheme showing the light-activation of cpFBPase via the
ferredoxin-thioredoxin system.

[0792]FIG. 12 is an alignment of cyFBPase polypeptides and cpFBPase
polypeptides. The polypeptide sequences were aligned using AlignX program
from Vector NTI suite (InforMax, Bethesda, Md.). Multiple alignment was
done with a gap opening penalty of 10 and a gap extension of 0.01. The
predicted chloroplastic transit peptide of the cpFBPase polypeptides is
boxed. The redox regulatory insertion and the cysteines involved in
disulfide bridge formation are indicated. Finally, the active site region
is also boxed, and the amino acid residues Asn237, Tyr269, Tyr289 and
Arg268 which bind the 6-phosphate of fructose-1,6-bisphosphate, and
Lys299 which binds the fructose are shown.

[0793]FIG. 13 shows a binary vector p1597, for increased expression in
Oryza sativa of a Chlamydomonas reindhardtii nucleic acid encoding a
cpFBPase polypeptide under the control of a GOS2 promoter (internal
reference PRO0129).

[0796]FIG. 16 represents the binary for vector endogenous gene silencing
in Oryza sativa using a SIK nucleic acid represented by SEQ ID NO: 1
using a hairpin construct under the control of a constitutive promoter,
GOS2.

[0797]FIG. 17 shows a binary vector for expression in Oryza sativa of a
SIK-encoding nucleic acid from Oryza sativa under the control of a GOS2
promoter.

[0800]FIG. 20 is an alignment taken from Henrikson et al., 2005 (Plant
Physiol, Vol. 139, pp. 509-518). The Leu Zip domains in all HDZip I and
II proteins are in identical positions, C terminal to the HD. The HDZip
domains of HDZip I and II proteins are similar to each other in sequence,
although a number of amino acid positions distinguish HDZip I from II.
The amino acid at position 46 is invariant within the HDZip I and II, but
distinct between the classes. Several other amino acids, e.g. the ones at
positions 6, 25, 29, 30, 58, and 61, are invariable within the HDZip II
and differ from HDZip I amino acids, which show variation at these
positions.

[0801]FIG. 21 shows a binary vector for increased expression in Oryza
sativa of the Arabidopsis thaliana Class II HD-Zip transcription factor
(HAT4)-encoding nucleic acid under the control of a GOS2 promoter.

[0802]FIG. 22 shows the domain structure of two examples of SYB1 proteins.
The Zinc finger domains are indicated in underlined bold.

[0804]FIG. 24 shows the binary vector for increased expression in Oryza
sativa of an Arabidopsis thaliana SYB1 protein-encoding nucleic acid
under the control of a GOS2 promoter (internal reference PRO0129).

[0805]FIG. 25 details examples of sequences useful in performing the
methods according to the present invention. SEQ ID NO: 1 to SEQ ID NO: 56
relate to AZ sequences. SEQ ID NO: 1 and 2 represent the AZ coding
sequence CDS3104 and the deduced protein sequence. SEQ ID NO: 53 and 47
represent the AZ coding sequence CDS3108 and the deduced protein
sequence. SEQ ID NO: 3 to 6 represent conserved sequences that may be
present in an AZ protein, SEQ ID NO: 9 and 55 represent sequences of the
WSI18 promoter used in the examples section. SEQ ID NO: 10 to 52 are
examples of sequences of other AZ proteins and nucleic acids encoding
these proteins, SEQ ID NO: 54 and 56 are the sequences of the rice GOS2
promoter.

[0806]SEQ ID NO: 56 to 153 relate to SYT sequences. SYT nucleic acid
sequences are presented from start to stop. The majority of these
sequences are derived from EST sequencing, which is of lower quality.
Therefore, nucleic acid substitutions may be encountered.

[0807]SEQ ID NO: 56, and SEQ ID NO: 154 to 208 represent examples of
sequences relating to cpFBPase, useful in performing the methods
according to the present invention.

[0808]SEQ ID NO: 56 and 209 to 228 are examples of sequences useful in
performing the methods according to the present invention relating to SIK
proteins/nucleic acids, or useful in isolating such sequences. Sequences
may result from public EST assemblies, with lesser quality sequencing. As
a consequence, a few nucleic acid substitutions may be expected. The 5'
and 3' UTR of the naturally transcribed sequences may also be used for
the performing the methods of the invention for the reduction or
substantial elimination of endogenous SIK gene expression.

[0809]SEQ ID NO: 229 to 284 are examples of sequences useful in performing
the methods according to the present invention relating to Class II
HD-Zip transcription factor (HAT4).

[0810]SEQ ID NO: 56, and SEQ ID NO: 285 to 347 are examples of sequences
relating to SYB1 proteins and nucleic acids and are useful in performing
the methods according to the present invention.

EXAMPLES

[0811]The present invention will now be described with reference to the
following examples, which are by way of illustration alone. The following
examples are not intended to completely define or otherwise limit the
scope of the invention.

[0812]DNA manipulation: unless otherwise stated, recombinant DNA
techniques are performed according to standard protocols described in
(Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold
Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of
Ausubel et al. (1994), Current Protocols in Molecular Biology, Current
Protocols. Standard materials and methods for plant molecular work are
described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy,
published by BIOS Scientific Publications Ltd (UK) and Blackwell
Scientific Publications (UK).

Example 1

Identification of Sequences Related to the Nucleic Acid Sequence used in
the Methods of the Invention

[0813]Sequences (full length cDNA, ESTs or genomic) related to the nucleic
acid sequence used in the methods of the present invention were
identified amongst those maintained in the Entrez Nucleotides database at
the National Center for Biotechnology Information (NCBI) using database
sequence search tools, such as the Basic Local Alignment Tool (BLAST)
(Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al.
(1997) Nucleic Acids Res. 25:3389-3402). The program is used to find
regions of local similarity between sequences by comparing nucleic acid
or polypeptide sequences to sequence databases and by calculating the
statistical significance of matches. For example, the polypeptide encoded
by the nucleic acid used in the present invention was used for the
TBLASTN algorithm, with default settings and the filter to ignore low
complexity sequences set off. The output of the analysis was viewed by
pairwise comparison, and ranked according to the probability score
(E-value), where the score reflect the probability that a particular
alignment occurs by chance (the lower the E-value, the more significant
the hit). In addition to E-values, comparisons were also scored by
percentage identity. Percentage identity refers to the number of
identical nucleotides (or amino acids) between the two compared nucleic
acid (or polypeptide) sequences over a particular length. In some
instances, the default parameters may be adjusted to modify the
stringency of the search. For example the E-value may be increased to
show less stringent matches. This way, short nearly exact matches may be
identified.

[0814]Table A provides a list of nucleic acid sequences related to the
nucleic acid sequence used in the methods of the present invention.

[0815]In some instances, related sequences have tentatively been assembled
and publicly disclosed by research institutions, such as The Institute
for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database
may be used to identify such related sequences, either by keyword search
or by using the BLAST algorithm with the nucleic acid or polypeptide
sequence of interest.

Example 2

Gene Cloning of AZ

[0816]The Arabidopsis AZ encoding gene (CDS3104) was amplified by PCR
using as template an Arabidopsis thaliana seedling cDNA library
(Invitrogen, Paisley, UK). After reverse transcription of RNA extracted
from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert
size of the bank was 1.5 kb, and the original number of clones was of
1.59×107 cfu. Original titer was determined to be
9.6×105 cfu/ml, after a first amplification of
6×1011 cfu/ml. After plasmid extraction, 200 ng of template
was used in a 50 μl PCR mix. Primers prm06717 (sense, AttB1 site in
italic, start codon in bold:
5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatgtgctgtggatcagacc-3') (SEQ ID NO
7) and prm06718 (reverse, complementary, AttB2 site in italic:
5'-ggggaccactttgtacaagaaagctgggtggttaggtctctcaattctgc-3') (SEQ ID NO 8),
which include the AttB sites for Gateway recombination, were used for PCR
amplification. PCR was performed using Hifi Taq DNA polymerase in
standard conditions. A PCR fragment of the expected size was amplified
and purified also using standard methods. The first step of the Gateway
procedure, the BP reaction, was then performed, during which the PCR
fragment recombines in vivo with the pDONR201 plasmid to produce,
according to the Gateway terminology, an "entry clone", p07. Plasmid
pDONR201 was purchased from Invitrogen, as part of the Gateway®
technology.

Example 3

Vector Construction

[0817]The entry clone p07 were subsequently used in an LR reaction with
p02417, a destination vector used for plant (Oryza sativa)
transformation. This vector contains as functional elements within the
T-DNA borders: a plant selectable marker; a screenable marker expression
cassette; and a Gateway cassette intended for LR in vivo recombination
with the sequence of interest already cloned in the entry clone. A rice
WSI18 promoter (SEQ ID NO: 9) for seed specific expression (PRO0151) was
located upstream of this Gateway cassette (p056, FIG. 4).

[0818]Many different binary (and super binary) vector systems have been
described for plant transformation (e.g. An, G. in Agrobacterium
Protocols. Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA
and MR Davey eds. Humana Press, Totowa, N.J.). Many are based on the
vector pBIN19 described by Bevan (Nucleic Acid Research. 1984.
12:8711-8721) that includes a plant gene expression cassette flanked by
the left and right border sequences from the Ti plasmid of Agrobacterium
tumefaciens. A plant gene expression cassette consists of at least two
genes--a selection marker gene and a plant promoter regulating the
transcription of the cDNA or genomic DNA of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.
Nos. 5,767,3666 and 6,225,105). Similarly, various promoters can be used
to regulate the trait gene to provide constitutive, developmental, tissue
or environmental regulation of gene transcription.

[0819]After the LR recombination step, the resulting expression vector,
p056 (FIG. 4), was transformed into Agrobacterium strain LBA4044 using
heat shock or electroporation protocols. Transformed colonies were grown
on YEP media and selected by respective antibiotics for two days at
28° C. These Agrobacterium cultures were used for the plant
transformation.

[0820]Other Agrobacterium tumefaciens strains can be used for plant
transformation and are well known in the art. Examples of such strains
are C58C1 or EHA105.

Example 4

Plant Transformation

Rice Transformation

[0821]The Agrobacterium containing the expression vector was used to
transform Oryza sativa plants. Mature dry seeds of the rice japonica
cultivar Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl2, followed by a 6 times 15 minutes wash with sterile distilled
water. The sterile seeds were then germinated on a medium containing
2,4-D (callus induction medium). After incubation in the dark for four
weeks, embryogenic, scutellum-derived calli were excised and propagated
on the same medium. After two weeks, the calli were multiplied or
propagated by subculture on the same medium for another 2 weeks.
Embryogenic callus pieces were sub-cultured on fresh medium 3 days before
co-cultivation (to boost cell division activity).

[0822]Agrobacterium strain LBA4404 containing the expression vector was
used for cocultivation. Agrobacterium was inoculated on AB medium with
the appropriate antibiotics and cultured for 3 days at 28° C. The
bacteria were then collected and suspended in liquid co-cultivation
medium to a density (OD600) of about 1. The suspension was then
transferred to a Petri dish and the calli immersed in the suspension for
15 minutes. The callus tissues were then blotted dry on a filter paper
and transferred to solidified, co-cultivation medium and incubated for 3
days in the dark at 25° C. Co-cultivated calli were grown on
2,4-D-containing medium for 4 weeks in the dark at 28° C. in the
presence of a selection agent. During this period, rapidly growing
resistant callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to five
weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks
on an auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse.

[0823]Approximately 35 independent T0 rice transformants were generated
for one construct. The primary transformants were transferred from a
tissue culture chamber to a greenhouse. After a quantitative PCR analysis
to verify copy number of the T-DNA insert, only single copy transgenic
plants that exhibit tolerance to the selection agent were kept for
harvest of T1 seed. Seeds were then harvested three to five months after
transplanting. The method yielded single locus transformants at a rate of
over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

[0824]Transformation of maize (Zea mays) is performed with a modification
of the method described by Ishida et al. (1996) Nature Biotech 14(6):
745-50. Transformation is genotype-dependent in corn and only specific
genotypes are amenable to transformation and regeneration. The inbred
line A188 (University of Minnesota) or hybrids with A188 as a parent are
good sources of donor material for tansformation, but other genotypes can
be used successfully as well. Ears are harvested from corn plant
approximately 11 days after pollination (DAP) when the length of the
immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated
with Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised embryos
are grown on callus induction medium, then maize regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to maize rooting medium and
incubated at 25° C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Wheat Transformation

[0825]Transformation of wheat is performed with the method described by
Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite
(available from CIMMYT, Mexico) is commonly used in transformation.
Immature embryos are co-cultivated with Agrobacterium tumefaciens
containing the expression vector, and transgenic plants are recovered
through organogenesis. After incubation with Agrobacterium, the embryos
are grown in vitro on callus induction medium, then regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and incubated
at 25° C. for 2-3 weeks, until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Soybean Transformation

[0826]Soybean is transformed according to a modification of the method
described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several
commercial soybean varieties are amenable to transformation by this
method. The cultivar Jack (available from the Illinois Seed foundation)
is commonly used for transformation. Soybean seeds are sterilised for in
vitro sowing. The hypocotyl, the radicle and one cotyledon are excised
from seven-day old young seedlings. The epicotyl and the remaining
cotyledon are further grown to develop axillary nodes. These axillary
nodes are excised and incubated with Agrobacterium tumefaciens containing
the expression vector. After the cocultivation treatment, the explants
are washed and transferred to selection media. Regenerated shoots are
excised and placed on a shoot elongation medium. Shoots no longer than 1
cm are placed on rooting medium until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Rapeseed/Canola Transformation

[0827]Cotyledonary petioles and hypocotyls of 5-6 day old young seedling
are used as explants for tissue culture and transformed according to
Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar
Westar (Agriculture Canada) is the standard variety used for
transformation, but other varieties can also be used. Canola seeds are
surface-sterilized for in vitro sowing. The cotyledon petiole explants
with the cotyledon attached are excised from the in vitro seedlings, and
inoculated with Agrobacterium (containing the expression vector) by
dipping the cut end of the petiole explant into the bacterial suspension.
The explants are then cultured for 2 days on MSBAP-3 medium containing 3
mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on
MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection
agent until shoot regeneration. When the shoots are 5-10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred
to the rooting medium (MS0) for root induction. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from plants
that exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.

Alfalfa Transformation

[0828]A regenerating clone of alfalfa (Medicago sativa) is transformed
using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847).
Regeneration and transformation of alfalfa is genotype dependent and
therefore a regenerating plant is required. Methods to obtain
regenerating plants have been described. For example, these can be
selected from the cultivar Rangelander (Agriculture Canada) or any other
commercial alfalfa variety as described by Brown DCW and A Atanassov
(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the
RA3 variety (University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants
are cocultivated with an overnight culture of Agrobacterium tumefaciens
C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing the expression vector. The explants are cocultivated for 3 d
in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and Skoog,
1962) and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to inhibit
Agrobacterium growth. After several weeks, somatic embryos are
transferred to BOi2Y development medium containing no growth regulators,
no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings were
transplanted into pots and grown in a greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Example 5

Evaluation Setup of AZ Expression in Rice under the Control of the Rice
WSI18 Promoter

[0829]Approximately 15 to 20 independent T0 rice transformants were
generated. The primary transformants were transferred from a tissue
culture chamber to a greenhouse for growing and harvest of T1 seed. Seven
events, of which the T1 progeny segregated 3:1 for presence/absence of
the transgene, were retained. For each of these events, approximately 10
T1 seedlings containing the transgene (hetero- and homo-zygotes) and
approximately 10 T1 seedlings lacking the transgene (nullizygotes) were
selected by monitoring visual marker expression. The selected T1 plants
were transferred to a greenhouse. Each plant received a unique barcode
label to link unambiguously the phenotyping data to the corresponding
plant. The selected T1 plants were grown on soil in 10 cm diameter,
specially designed pots with transparent bottoms to allow visualization
of the roots, under the following environmental settings:
photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime
temperature=28° C., night time temperature=22° C., relative
humidity=60-70%. Transgenic plants and the corresponding nullizygotes
were grown side-by-side at random positions. Care was taken that the
plants were not subjected to any stress. From the stage of sowing until
the stage of maturity the plants were passed several times through a
digital imaging cabinet. At each time point digital images
(2048×1536 pixels, 16 million colours) were taken of each plant
from at least 6 different angles. A digital camera also recorded images
through the bottom of the pot during plant growth.

Drought Screen

[0830]Plants from T2 seeds are grown in potting soil under normal
conditions until they approached the heading stage. They are then
transferred to a "dry" section where irrigation is withheld. Humidity
probes are inserted in randomly chosen pots to monitor the soil water
content (SWC). When SWC goes below certain thresholds, the plants are
automatically re-watered continuously until a normal level was reached
again. The plants are then re-transferred again to normal conditions. The
rest of the cultivation (plant maturation, seed harvest) is the same as
for plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

Nitrogen use Efficiency Screen

[0831]Rice plants from T2 seeds are grown in potting soil under normal
conditions except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

[0832]The plant aboveground area (or leafy biomass) was determined by
counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This value was
averaged for the pictures taken on the same time point from the different
angles and was converted to a physical surface value expressed in square
mm by calibration. Experiments show that the aboveground plant area
measured this way correlates with the biomass of plant parts above
ground. The above ground area is the time point at which the plant had
reached its maximal leafy biomass. Root features such as total projected
area (which can be correlated to total root volume), average diameter and
length of roots above a certain thickness threshold (length of thick
roots, or length of thin roots) were deduced from the generated image
using appropriate software.

[0833]The mature primary panicles were harvested, bagged, barcode-labelled
and then dried for three days in the oven at 37° C. The panicles
were then threshed and all the seeds collected. The filled husks were
separated from the empty ones using an air-blowing device. After
separation, both seed lots were then counted using a commercially
available counting machine. The empty husks were discarded. The filled
husks were weighed on an analytical balance and the cross-sectional area
of the seeds was measured using digital imaging. This procedure resulted
in the set of the following seed-related parameters:

[0834]The number of filled seeds was determined by counting the number of
filled husks that remained after the separation step. The total seed
yield (total seed weight) was measured by weighing all filled husks
harvested from a plant. Total seed number per plant was measured by
counting the number of husks harvested from a plant. The harvest index
(HI) in the present invention is defined as the ratio between the total
seed yield and the above ground area (mm2), multiplied by a factor
106. Thousand Kernel Weight (TKW) is extrapolated from the number of
filled seeds counted and their total weight. The seed fill rate as
defined in the present invention is the proportion (expressed as a %) of
the number of filled seeds over the total number of seeds (or florets).
These parameters were derived in an automated way from the digital images
using image analysis software and were analysed statistically. Individual
seed parameters (including width, length, area, weight) were measured
using a custom-made device consisting of two main components, a weighing
and imaging device, coupled to software for image analysis.

[0835]A two factor ANOVA (analyses of variance) corrected for the
unbalanced design was used as statistical model for the overall
evaluation of plant phenotypic characteristics. An F-test was carried out
on all the parameters measured of all the plants of all the events
transformed with that gene. The F-test was carried out to check for an
effect of the gene over all the transformation events and to verify for
an overall effect of the gene, also named herein "global gene effect". If
the value of the F test shows that the data are significant, than it is
concluded that there is a "gene" effect, meaning that not only presence
or the position of the gene is causing the effect. The threshold for
significance for a true global gene effect is set at 5% probability level
for the F test.

[0836]To check for an effect of the genes within an event, i.e., for a
line-specific effect, a t-test was performed within each event using data
sets from the transgenic plants and the corresponding null plants. "Null
plants" or "null segregants" or "nullizygotes" are the plants treated in
the same way as the transgenic plant, but from which the transgene has
segregated. Null plants can also be described as the homozygous negative
transformed plants. The threshold for significance for the t-test is set
at 10% probability level. The results for some events can be above or
below this threshold. This is based on the hypothesis that a gene might
only have an effect in certain positions in the genome, and that the
occurrence of this position-dependent effect is not uncommon. This kind
of gene effect is also named herein a "line effect of the gene". The
p-value is obtained by comparing the t-value to the t-distribution or
alternatively, by comparing the F-value to the F-distribution. The
p-value then gives the probability that the null hypothesis (i.e., that
there is no effect of the transgene) is correct.

[0837]The data obtained for AZ in the first experiment were confirmed in a
second experiment with T2 plants. Four lines that had the correct
expression pattern were selected for further analysis. Seed batches from
the positive plants (both hetero- and homozygotes) in T1, were screened
by monitoring marker expression. For each chosen event, the heterozygote
seed batches were then retained for T2 evaluation. Within each seed batch
an equal number of positive and negative plants were grown in the
greenhouse for evaluation.

[0838]A total number of 120 AZ transformed plants were evaluated in the T2
generation, that is 30 plants per event of which 15 positives for the
transgene, and 15 negatives.

[0839]Because two experiments with overlapping events had been carried
out, a combined analysis was performed. This is useful to check
consistency of the effects over the two experiments, and if this is the
case, to accumulate evidence from both experiments in order to increase
confidence in the conclusion. The method used was a mixed-model approach
that takes into account the multilevel structure of the data (i.e.
experiment--event--segregants). P-values are obtained by comparing
likelihood ratio test to chi square distributions.

[0840]Upon analysis of the seeds as described above, the inventors found
that plants transformed with the AZ gene construct had a higher seed
yield, expressed as thousand kernel weight, compared to plants lacking
the AZ transgene. Furthermore, increased emergence vigour and increased
greenness index was observed in plants carrying the transgene compared to
the control plants.

[0841]For one of the constructs, thousand-kernel weight was increased with
2.7% in the T1 generation. These positive results were again obtained in
the T2 generation (increase of 2.1%). The T2 data were re-evaluated in a
combined analysis with the results for the T1 generation, and the
obtained p-values showed that the observed effects were highly
significant.

Example 7

Gene Cloning of AtSYT1

[0842]The Arabidopsis thaliana AtSYT1 gene was amplified by PCR using as
template an Arabidopsis thaliana seedling cDNA library (Invitrogen,
Paisley, UK). After reverse transcription of RNA extracted from
seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size
of the bank was 1.5 kb and the original number of clones was of the order
of 1.59×107 cfu. Original titer was determined to be
9.6×105 cfu/ml after first amplification of 6×1011
cfu/ml. After plasmid extraction, 200 ng of template was used in a 50
μl PCR mix. Primers prm06681 (SEQ ID NO: 148; sense, start codon in
bold, AttB1 site in italic: 5'-GGGGACAAGTTTG TA
CAAAAAAGCAGGCTTAAACAATGCAACAGCACCTGATG -3') and prm06682 (SEQ ID NO: 149;
reverse, complementary, AttB2 site in italic:
5'-GGGGACCACTTTGTACAAGAAAGCTGGG TCATCATTAAGATTCCTTGTG C-3'), which
include the AttB sites for Gateway recombination, were used for PCR
amplification. PCR was performed using Hifi Taq DNA polymerase in
standard conditions. A PCR fragment of 727 by (including attB sites) was
amplified and purified also using standard methods. The first step of the
Gateway procedure, the BP reaction, was then performed, during which the
PCR fragment recombines in vivo with the pDONR201 plasmid to produce,
according to the Gateway terminology, an "entry clone", p07466. Plasmid
pDONR201 was purchased from Invitrogen, as part of the Gateway®
technology.

Example 8

Vector Construction

[0843]The entry clone p07466 was subsequently used in an LR reaction with
p00640, a destination vector used for plant (Oryza sativa)
transformation. This vector contains as functional elements within the
T-DNA borders: a plant selectable marker; a screenable marker expression
cassette; and a Gateway cassette intended for LR in vivo recombination
with the sequence of interest already cloned in the entry clone. A rice
GOS2 promoter (SEQ ID NO: 145) for constitutive expression (PRO0129) was
located upstream of this Gateway cassette.

[0844]Many different binary (and super binary) vector systems have been
described for plant transformation (e.g. An, G. in Agrobacterium
Protocols. Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA
and MR Davey eds. Humana Press, Totowa, N.J.). Many are based on the
vector pBIN19 described by Bevan (Nucleic Acid Research. 1984.
12:8711-8721) that includes a plant gene expression cassette flanked by
the left and right border sequences from the Ti plasmid of Agrobacterium
tumefaciens. A plant gene expression cassette consists of at least two
genes--a selection marker gene and a plant promoter regulating the
transcription of the cDNA or genomic DNA of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.
Nos. 5,767,3666 and 6,225,105). Similarly, various promoters can be used
to regulate the trait gene to provide constitutive, developmental, tissue
or environmental regulation of gene transcription.

[0845]After the LR recombination step, the resulting expression vector
pGOS2::AtSYT1 (FIG. 9) was transformed into Agrobacterium strain LBA4044
using heat shock or electroporation protocols. Transformed colonies were
grown on YEP media and selected by respective antibiotics for two days at
28° C. These Agrobacterium cultures were used for the plant
transformation.

[0846]Other Agrobacterium tumefaciens strains can be used for plant
transformation and are well known in the art. Examples of such strains
are C58C1 or EHA105.

Example 9

Plant Transformation

Rice Transformation

[0847]The Agrobacterium containing the expression vector was used to
transform Oryza sativa plants. Mature dry seeds of the rice japonica
cultivar Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl2, followed by a 6 times 15 minutes wash with sterile distilled
water. The sterile seeds were then germinated on a medium containing
2,4-D (callus induction medium). After incubation in the dark for four
weeks, embryogenic, scutellum-derived calli were excised and propagated
on the same medium. After two weeks, the calli were multiplied or
propagated by subculture on the same medium for another 2 weeks.
Embryogenic callus pieces were sub-cultured on fresh medium 3 days before
co-cultivation (to boost cell division activity).

[0848]Agrobacterium strain LBA4404 containing the expression vector was
used for cocultivation. Agrobacterium was inoculated on AB medium with
the appropriate antibiotics and cultured for 3 days at 28° C. The
bacteria were then collected and suspended in liquid co-cultivation
medium to a density (OD600) of about 1. The suspension was then
transferred to a Petri dish and the calli immersed in the suspension for
15 minutes. The callus tissues were then blotted dry on a filter paper
and transferred to solidified, co-cultivation medium and incubated for 3
days in the dark at 25° C. Co-cultivated calli were grown on
2,4-D-containing medium for 4 weeks in the dark at 28° C. in the
presence of a selection agent. During this period, rapidly growing
resistant callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to five
weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks
on an auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse.

[0849]Approximately 35 independent TO rice transformants were generated
for one construct. The primary transformants were transferred from a
tissue culture chamber to a greenhouse. After a quantitative PCR analysis
to verify copy number of the T-DNA insert, only single copy transgenic
plants that exhibit tolerance to the selection agent were kept for
harvest of T1 seed. Seeds were then harvested three to five months after
transplanting. The method yielded single locus transformants at a rate of
over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

[0850]Transformation of maize (Zea mays) is performed with a modification
of the method described by Ishida et al. (1996) Nature Biotech 14(6):
745-50. Transformation is genotype-dependent in corn and only specific
genotypes are amenable to transformation and regeneration. The inbred
line A188 (University of Minnesota) or hybrids with A188 as a parent are
good sources of donor material for tansformation, but other genotypes can
be used successfully as well. Ears are harvested from corn plant
approximately 11 days after pollination (DAP) when the length of the
immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated
with Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised embryos
are grown on callus induction medium, then maize regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to maize rooting medium and
incubated at 25° C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Wheat Transformation

[0851]Transformation of wheat is performed with the method described by
Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite
(available from CIMMYT, Mexico) is commonly used in transformation.
Immature embryos are co-cultivated with Agrobacterium tumefaciens
containing the expression vector, and transgenic plants are recovered
through organogenesis. After incubation with Agrobacterium, the embryos
are grown in vitro on callus induction medium, then regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and incubated
at 25° C. for 2-3 weeks, until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Soybean Transformation

[0852]Soybean is transformed according to a modification of the method
described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several
commercial soybean varieties are amenable to transformation by this
method. The cultivar Jack (available from the Illinois Seed foundation)
is commonly used for transformation. Soybean seeds are sterilised for in
vitro sowing. The hypocotyl, the radicle and one cotyledon are excised
from seven-day old young seedlings. The epicotyl and the remaining
cotyledon are further grown to develop axillary nodes. These axillary
nodes are excised and incubated with Agrobacterium tumefaciens containing
the expression vector. After the cocultivation treatment, the explants
are washed and transferred to selection media. Regenerated shoots are
excised and placed on a shoot elongation medium. Shoots no longer than 1
cm are placed on rooting medium until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

[0853]Rapeseed/Canola Transformation

[0854]Cotyledonary petioles and hypocotyls of 5-6 day old young seedling
are used as explants for tissue culture and transformed according to
Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar
Westar (Agriculture Canada) is the standard variety used for
transformation, but other varieties can also be used. Canola seeds are
surface-sterilized for in vitro sowing. The cotyledon petiole explants
with the cotyledon attached are excised from the in vitro seedlings, and
inoculated with Agrobacterium (containing the expression vector) by
dipping the cut end of the petiole explant into the bacterial suspension.
The explants are then cultured for 2 days on MSBAP-3 medium containing 3
mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on
MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection
agent until shoot regeneration. When the shoots are 5-10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred
to the rooting medium (MSO) for root induction. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from plants
that exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.

Alfalfa Transformation

[0855]A regenerating clone of alfalfa (Medicago sativa) is transformed
using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847).
Regeneration and transformation of alfalfa is genotype dependent and
therefore a regenerating plant is required. Methods to obtain
regenerating plants have been described. For example, these can be
selected from the cultivar Rangelander (Agriculture Canada) or any other
commercial alfalfa variety as described by Brown DCW and A Atanassov
(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the
RA3 variety (University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants
are cocultivated with an overnight culture of Agrobacterium tumefaciens
C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing the expression vector. The explants are cocultivated for 3 d
in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and Skoog,
1962) and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to inhibit
Agrobacterium growth. After several weeks, somatic embryos are
transferred to BOi2Y development medium containing no growth regulators,
no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings were
transplanted into pots and grown in a greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

[0856]Four events, of which the T1 progeny segregated 3:1 for
presence/absence of the transgene, were retained. For each of these
events, approximately 15 T1 seedlings containing the transgene (hetero-
and homo-zygotes) and approximately 15 T1 seedlings lacking the transgene
(nullizygotes) were selected by monitoring visual marker expression. The
transgenic plants and the corresponding nullizygotes were grown
side-by-side at random positions. Greenhouse conditions were of shorts
days (12 hours light), with temperatures on average 28° C. in the
light and 22° C. in the dark, and a relative humidity on average
of 70%.

Salt Stress Screen

[0857]Plants were grown on a substrate made of coco fibers and argex (3 to
1 ratio). A normal nutrient solution was used during the first two weeks
after transplanting the plantlets in the greenhouse. After the first two
weeks, 25 mM of salt (NaCl) was added to the nutrient solution, until the
plants were harvested. Seed-related parameters were then measured.

Drought Screen

[0858]Plants are grown in potting soil under normal conditions until they
approach the heading stage. They are then transferred to a "dry" section
where irrigation is withheld. Humidity probes are inserted in randomly
chosen pots to monitor the soil water content (SWC). When SWC goes below
certain thresholds, the plants are automatically re-watered continuously
until a normal level is reached again. The plants are then re-transferred
again to normal conditions. The rest of the cultivation (plant
maturation, seed harvest) is the same as for plants not grown under
abiotic stress conditions. Seed-related parameters are then measured An
alternative method to impose water stress on the transgenic plants is by
treatment with water containing an osmolyte such as polyethylene glycol
(PEG) at specific water potential.

[0859]Since PEG may be toxic, the plants are given only a short-term
exposure and then normal watering is resumed.

Reduced Nutrient (Nitrogen) Availability Screen

[0860]The rice plants are grown in potting soil under normal conditions
except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Seed-related
parameters are then measured

Statistical Analysis: F-Test

[0861]A two factor ANOVA (analysis of variants) was used as a statistical
model for the overall evaluation of plant phenotypic characteristics. An
F-test was carried out on all the parameters measured of all the plants
of all the events transformed with the gene of the present invention. The
F-test was carried out to check for an effect of the gene over all the
transformation events and for an overall effect of the gene, also known
as a global gene effect. The threshold for significance for a true global
gene effect was set at a 5% probability level for the F-test. A
significant F-test value points to a gene effect, meaning that it is not
only the presence or position of the gene that is causing the differences
in phenotype.

Biomass-Related Parameter Measurement

[0862]From the stage of sowing until the stage of maturity the plants were
passed several times through a digital imaging cabinet. At each time
point digital images (2048×1536 pixels, 16 million colours) were
taken of each plant from at least 6 different angles.

[0863]The plant aboveground area (or leafy biomass) was determined by
counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This value was
averaged for the pictures taken on the same time point from the different
angles and was converted to a physical surface value expressed in square
mm by calibration. Experiments show that the aboveground plant area
measured this way correlates with the biomass of plant parts above
ground. The above ground area is the time point at which the plant had
reached its maximal leafy biomass. The early vigour is the plant
(seedling) aboveground area three weeks post-germination.

Seed-Related Parameter Measurements

[0864]The mature primary panicles were harvested, counted, bagged,
barcode-labeled and then dried for three days in an oven at 37° C.
The panicles were then threshed and all the seeds were collected and
counted. The filled husks were separated from the empty ones using an
air-blowing device. The empty husks were discarded and the remaining
fraction was counted again. The filled husks were weighed on an
analytical balance. The number of filled seeds was determined by counting
the number of filled husks that remained after the separation step. The
total seed yield was measured by weighing all filled husks harvested from
a plant. Total seed number per plant was measured by counting the number
of husks harvested from a plant. Thousand kernel weight (TKW) is
extrapolated from the number of filled seeds counted and their total
weight. The harvest index (HI) in the present invention is defined as the
ratio between the total seed yield and the above ground area (mm2),
multiplied by a factor 106. The total number of flowers per panicle
as defined in the present invention is the ratio between the total number
of seeds and the number of mature primary panicles. The seed fill rate as
defined in the present invention is the proportion (expressed as a %) of
the number of filled seeds over the total number of seeds (or florets).

[0865]The results of the evaluation of AtSYT1 transgenic rice plants
submitted to salt stress are presented in Table B. The percentage
difference between the transgenics and the corresponding nullizygotes is
also shown, with a P value from the F test below 0.05.

[0866]Aboveground biomass, early vigour, total seed yield, number of
filled seeds, seed fill rate, TKW and harvest index are significantly
increased in the AtSYT1 transgenic plants compared to the control plants
(in this case, the nullizygotes), under abiotic stress.

[0867]Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO:
154 and SEQ ID NO: 155 were identified amongst those maintained in the
Entrez Nucleotides database at the National Center for Biotechnology
Information (NCBI) using database sequence search tools, such as the
Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.
215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
The program is used to find regions of local similarity between sequences
by comparing nucleic acid or polypeptide sequences to sequence databases
and by calculating the statistical significance of matches. The
polypeptide encoded by SEQ ID NO: 154 was used for the TBLASTN algorithm,
with default settings and the filter to ignore low complexity sequences
set off. The output of the analysis was viewed by pairwise comparison,
and ranked according to the probability score (E-value), where the score
reflects the probability that a particular alignment occurs by chance
(the lower the E-value, the more significant the hit). In addition to
E-values, comparisons were also scored by percentage identity. Percentage
identity refers to the number of identical nucleotides (or amino acids)
between the two compared nucleic acid (or polypeptide) sequences over a
particular length. In some instances, the default parameters may be
adjusted to modify the stringency of the search.

[0868]In addition to the publicly available nucleic acid sequences
available at NCBI, proprietary sequence databases are also searched
following the same procedure as described herein above.

[0870]AlignX from the Vector NTI (Invitrogen) is based on the popular
Clustal algorithm of progressive alignment (Thompson et al. (1997)
Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res
31:3497-3500). A phylogenetic tree can be constructed using a
neighbour-joining clustering algorithm. Default values are for the gap
open penalty of 10, for the gap extension penalty of 0.1 and the selected
weight matrix is Blosum 62 (if polypeptides are aligned).

[0871]The result of the multiple sequence alignment using polypeptides
relevant in identifying the ones useful in performing the methods of the
invention is shown in FIG. 12 of the present application. Chloroplastic
FBPase polypeptides (cpFBPase) differ from cytoplasmic FBPase (cyFBPase)
polypeptides by the presence of a transit peptide at their N-terminus for
plastidic (chloroplastic) subcellular targeting (boxed in the figure). In
addition, the former contain an insertion of amino acids (also boxed, and
named redox regulatory insertion) comprising at least two cysteine
residues necessary for disulphide bridge formation (i.e. redox
regulation). The conserved cysteines are named after their position in
the mature pea (Pisum sativa) cpFBPase polypeptide, i.e. Cys153, Cys173
and Cys178. Cys153 and Cys173 usually are the two partners involved in
disulphide bridge formation (Chiadmi et al. (1999) EMBO J 18(23):
6809-6815). The active site motif of cpFBPase as defined in the Prosite
database is PS00124 and corresponds to the following amino acid sequence:
[AG]-[RK]-[LI]-X(1,2)-[LIV]-[FY]-E-X(2)-P-[LIVM]-[GSA], wherein [RK] is
the active site that binds and X any amino acid. The predicted active
site motif and predicted active site itself (comprised within the motif)
have also been boxed in FIG. 12. Also identified in FIG. 12 are amino
acid residues Asn237, Tyr269, Tyr289 and Arg268 which bind the
6-phosphate of fructose-1,6-bisphosphate, and Lys299 which binds the
fructose (Chiadmi et al. (1999) EMBO J 18(23): 6809-6815). These latter
are also numbered after their position in the mature pea (Pisum sativa)
cpFBPase polypeptide.

Example 14

Calculation of Global Percentage Identity between Polypeptide Sequences
Useful in Performing the Methods of the Invention

[0872]Global percentages of similarity and identity between full length
polypeptide sequences useful in performing the methods of the invention
were determined using one of the methods available in the art, the MatGAT
(Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29.
MatGAT: an application that generates similarity/identity matrices using
protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software
hosted by Ledion Bitincka). MatGAT software generates similarity/identity
matrices for DNA or protein sequences without needing pre-alignment of
the data. The program performs a series of pair-wise alignments using the
Myers and Miller global alignment algorithm (with a gap opening penalty
of 12, and a gap extension penalty of 2), calculates similarity and
identity using for example Blosum 62 (for polypeptides), and then places
the results in a distance matrix. Sequence similarity is shown in the
bottom half of the dividing line and sequence identity is shown in the
top half of the diagonal dividing line.

[0876]Results of the software analysis are shown in Table D for the global
similarity and identity over the full length of the polypeptide sequences
(excluding the partial polypeptide sequences). Percentage identity is
given above the diagonal and percentage similarity is given below the
diagonal.

[0877]The percentage identity between the polypeptide sequences useful in
performing the methods of the invention can be as low as 40% amino acid
identity compared to SEQ ID NO: 155.

Identification of Domains Comprised in Polypeptide Sequences useful in
Performing the Methods of the Invention

[0878]The Integrated Resource of Protein Families, Domains and Sites
(InterPro) database is an integrated interface for the commonly used
signature databases for text- and sequence-based searches. The InterPro
database combines these databases, which use different methodologies and
varying degrees of biological information about well-characterized
proteins to derive protein signatures. Collaborating databases include
SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs.
Interpro is hosted at the European Bioinformatics Institute in the United
Kingdom.

[0879]The results of the InterPro scan of the polypeptide sequence as
represented by SEQ ID NO: 155 are presented in Table E.

[0880]TargetP 1.1 predicts the subcellular location of eukaryotic
proteins. The location assignment is based on the predicted presence of
any of the N-terminal pre-sequences: chloroplast transit peptide (cTP),
mitochondrial targeting peptide (mTP) or secretory pathway signal peptide
(SP). Scores on which the final prediction is based are not really
probabilities, and they do not necessarily add to one. However, the
location with the highest score is the most likely according to TargetP,
and the relationship between the scores (the reliability class) may be an
indication of how certain the prediction is. The reliability class (RC)
ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP
is maintained at the server of the Technical University of Denmark.

[0881]For the sequences predicted to contain an N-terminal presequence a
potential cleavage site can also be predicted.

[0882]A number of parameters were selected, such as organism group
(non-plant or plant), cutoff sets (none, predefined set of cutoffs, or
user-specified set of cutoffs), and the calculation of prediction of
cleavage sites (yes or no).

[0883]The results of TargetP 1.1 analysis of the polypeptide sequence as
represented by SEQ ID NO: 155 are presented Table F. The "plant" organism
group has been selected, no cutoffs defined, and the predicted length of
the transit peptide requested. The subcellular localization of the
polypeptide sequence as represented by SEQ ID NO: 155 is the chloroplast,
and the predicted length of the transit peptide is of 56 amino acids
starting from the N-terminus (not as reliable as the prediction of the
subcellular localization itself, may vary in length of a few amino
acids). The mature pea cpFBPase has a transit peptide of 53 amino acids
in length. When aligning the pea cpFBPase and the cpFBPase of SEQ ID NO:
155, it is possible to deduce the length of the transit peptide in the
latter, also of 53 amino acids.

[0884]Many other algorithms can be used to perform such analyses,
including: [0885]ChloroP 1.1 hosted on the server of the Technical
University of Denmark; [0886]Protein Prowler Subcellular Localisation
Predictor version 1.2 hosted on the server of the Institute for Molecular
Bioscience, University of Queensland, Brisbane, Australia; [0887]PENCE
Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of
Alberta, Edmonton, Alberta, Canada;

Example 17

Assay Related to the Polypeptide Sequences useful in Performing the
Methods of the Invention

[0888]Polypeptide sequence as represented by SEQ ID NO: 155 is an enzyme
with as Enzyme Commission (EC; classification of enzymes by the reactions
they catalyse) number EC 3.1.3.11 for fructose-bisphosphatase (also
called D-fructose-1,6-bisphosphate 1-phosphohydrolase). The functional
assay maybe an assay for FBPase activity based on a colorimetric Pi
assay, as described by Huppe and Buchanan (1989) in Naturforsch. 44c:
487-494. Other methods to assay the enzymatic activity are described by
Alscher-Herman (1982) in Plant Physiol 70: 728-734.

Example 18

Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO: 154

[0889]Unless otherwise stated, recombinant DNA techniques are performed
according to standard protocols described in (Sambrook (2001) Molecular
Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory
Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994),
Current Protocols in Molecular Biology, Current Protocols. Standard
materials and methods for plant molecular work are described in Plant
Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific Publications
(UK).

[0890]The nucleic acid sequence used in the methods of the invention was
amplified by PCR using as template a Chlamydomonas reinhardtii CC-1690
cDNA library ("Core Library") (in Lambda ZAP II vector from Stratagene)
purchased at the Chlamy Center (formerly the Chlamydomonas Genetics
Center) at Duke University, North Carolina, USA. PCR was performed using
Hifi Taq DNA polymerase in standard conditions, using 200 ng of template
in a 50 μl PCR mix. The primers used were [0891]prm08448 SEQ ID NO:
206; sense, AttB1 site in lower case:
5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatggccgccaccatg-3'; and
[0892]prm08449 (SEQ ID NO: 207; reverse, complementary, AttB2 site in
lower case:
5'-ggggaccactttgtacaagaaagctgggtagctgcttagtgcttcttggt-3',which include
the AttB sites for Gateway recombination. The amplified PCR fragment was
purified also using standard methods. The first step of the Gateway
procedure, the BP reaction, was then performed, during which the PCR
fragment recombines in vivo with the pDONR201 plasmid to produce,
according to the Gateway terminology, an "entry clone", p15972. Plasmid
pDONR201 was purchased from Invitrogen, as part of the Gateway®
technology.

[0893]The entry clone p15972 was subsequently used in an LR reaction with
p05050, a destination vector used for Oryza sativa transformation. This
vector contains as functional elements within the T-DNA borders: a plant
selectable marker; a screenable marker expression cassette; and a Gateway
cassette intended for LR in vivo recombination with the nucleic acid
sequence of interest already cloned in the entry clone. A rice GOS2
promoter (SEQ ID NO: 208) for constitutive expression (PRO0129) was
located upstream of this Gateway cassette.

[0894]After the LR recombination step, the resulting expression vector
pGOS2::FBPase (FIG. 13) was transformed into Agrobacterium strain LBA4044
according to methods well known in the art.

Example 20

Plant Transformation

Rice Transformation

[0895]The Agrobacterium containing the expression vector was used to
transform Oryza sativa plants. Mature dry seeds of the rice japonica
cultivar Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl2, followed by a 6 times 15 minutes wash with sterile distilled
water. The sterile seeds were then germinated on a medium containing
2,4-D (callus induction medium). After incubation in the dark for four
weeks, embryogenic, scutellum-derived calli were excised and propagated
on the same medium. After two weeks, the calli were multiplied or
propagated by subculture on the same medium for another 2 weeks.
Embryogenic callus pieces were sub-cultured on fresh medium 3 days before
co-cultivation (to boost cell division activity).

[0896]Agrobacterium strain LBA4404 containing the expression vector was
used for cocultivation. Agrobacterium was inoculated on AB medium with
the appropriate antibiotics and cultured for 3 days at 28° C. The
bacteria were then collected and suspended in liquid co-cultivation
medium to a density (OD600) of about 1. The suspension was then
transferred to a Petri dish and the calli immersed in the suspension for
15 minutes. The callus tissues were then blotted dry on a filter paper
and transferred to solidified, co-cultivation medium and incubated for 3
days in the dark at 25° C. Co-cultivated calli were grown on
2,4-D-containing medium for 4 weeks in the dark at 28° C. in the
presence of a selection agent. During this period, rapidly growing
resistant callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to five
weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks
on an auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse.

[0897]Approximately 35 independent T0 rice transformants were generated
for one construct. The primary transformants were transferred from a
tissue culture chamber to a greenhouse. After a quantitative PCR analysis
to verify copy number of the T-DNA insert, only single copy transgenic
plants that exhibit tolerance to the selection agent were kept for
harvest of T1 seed. Seeds were then harvested three to five months after
transplanting. The method yielded single locus transformants at a rate of
over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

[0898]Transformation of maize (Zea mays) is performed with a modification
of the method described by Ishida et al. (1996) Nature Biotech 14(6):
745-50. Transformation is genotype-dependent in corn and only specific
genotypes are amenable to transformation and regeneration. The inbred
line A188 (University of Minnesota) or hybrids with A188 as a parent are
good sources of donor material for tansformation, but other genotypes can
be used successfully as well. Ears are harvested from corn plant
approximately 11 days after pollination (DAP) when the length of the
immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated
with Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised embryos
are grown on callus induction medium, then maize regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to maize rooting medium and
incubated at 25° C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Wheat Transformation

[0899]Transformation of wheat is performed with the method described by
Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite
(available from CIMMYT, Mexico) is commonly used in transformation.
Immature embryos are co-cultivated with Agrobacterium tumefaciens
containing the expression vector, and transgenic plants are recovered
through organogenesis. After incubation with Agrobacterium, the embryos
are grown in vitro on callus induction medium, then regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and incubated
at 25° C. for 2-3 weeks, until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Soybean Transformation

[0900]Soybean is transformed according to a modification of the method
described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several
commercial soybean varieties are amenable to transformation by this
method. The cultivar Jack (available from the Illinois Seed foundation)
is commonly used for transformation. Soybean seeds are sterilised for in
vitro sowing. The hypocotyl, the radicle and one cotyledon are excised
from seven-day old young seedlings. The epicotyl and the remaining
cotyledon are further grown to develop axillary nodes. These axillary
nodes are excised and incubated with Agrobacterium tumefaciens containing
the expression vector. After the cocultivation treatment, the explants
are washed and transferred to selection media. Regenerated shoots are
excised and placed on a shoot elongation medium. Shoots no longer than 1
cm are placed on rooting medium until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Rapeseed/Canola Transformation

[0901]Cotyledonary petioles and hypocotyls of 5-6 day old young seedling
are used as explants for tissue culture and transformed according to
Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar
Westar (Agriculture Canada) is the standard variety used for
transformation, but other varieties can also be used. Canola seeds are
surface-sterilized for in vitro sowing. The cotyledon petiole explants
with the cotyledon attached are excised from the in vitro seedlings, and
inoculated with Agrobacterium (containing the expression vector) by
dipping the cut end of the petiole explant into the bacterial suspension.
The explants are then cultured for 2 days on MSBAP-3 medium containing 3
mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on
MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection
agent until shoot regeneration. When the shoots are 5-10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred
to the rooting medium (MS0) for root induction. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from plants
that exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.

Alfalfa Transformation

[0902]A regenerating clone of alfalfa (Medicago sativa) is transformed
using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847).
Regeneration and transformation of alfalfa is genotype dependent and
therefore a regenerating plant is required. Methods to obtain
regenerating plants have been described. For example, these can be
selected from the cultivar Rangelander (Agriculture Canada) or any other
commercial alfalfa variety as described by Brown D C W and A Atanassov
(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the
RA3 variety (University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants
are cocultivated with an overnight culture of Agrobacterium tumefaciens
C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing the expression vector. The explants are cocultivated for 3 d
in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and Skoog,
1962) and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to inhibit
Agrobacterium growth. After several weeks, somatic embryos are
transferred to BOi2Y development medium containing no growth regulators,
no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings were
transplanted into pots and grown in a greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Example 21

Phenotypic Evaluation Procedure

21.1 Evaluation Setup

[0903]Approximately 35 independent T0 rice transformants were generated.
The primary transformants were transferred from a tissue culture chamber
to a greenhouse for growing and harvest of T1 seed. Eight events, of
which the T1 progeny segregated 3:1 for presence/absence of the
transgene, were retained. For each of these events, approximately 10 T1
seedlings containing the transgene (hetero- and homo-zygotes) and
approximately 10 T1 seedlings lacking the transgene (nullizygotes) were
selected by monitoring visual marker expression. The transgenic plants
and the corresponding nullizygotes were grown side-by-side at random
positions. Greenhouse conditions were of shorts days (12 hours light),
28° C. in the light and 22° C. in the dark, and a relative
humidity of 70%.

Drought Screen

[0904]Plants from T2 seeds are grown in potting soil under normal
conditions until they approached the heading stage. They are then
transferred to a "dry" section where irrigation is withheld. Humidity
probes are inserted in randomly chosen pots to monitor the soil water
content (SWC). When SWC goes below certain thresholds, the plants are
automatically re-watered continuously until a normal level was reached
again. The plants are then re-transferred again to normal conditions. The
rest of the cultivation (plant maturation, seed harvest) is the same as
for plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

Nitrogen use Efficiency Screen

[0905]Rice plants from T2 seeds are grown in potting soil under normal
conditions except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

[0906]Four T1 events were further evaluated in the T2 generation following
the same evaluation procedure as for the T1 generation but with more
individuals per event. From the stage of sowing until the stage of
maturity the plants were passed several times through a digital imaging
cabinet. At each time point digital images (2048×1536 pixels, 16
million colours) were taken of each plant from at least 6 different
angles.

21.2 Statistical Analysis: F-Test

[0907]A two factor ANOVA (analysis of variants) was used as a statistical
model for the overall evaluation of plant phenotypic characteristics. An
F-test was carried out on all the parameters measured of all the plants
of all the events transformed with the gene of the present invention. The
F-test was carried out to check for an effect of the gene over all the
transformation events and to verify for an overall effect of the gene,
also known as a global gene effect. The threshold for significance for a
true global gene effect was set at a 5% probability level for the F-test.
A significant F-test value points to a gene effect, meaning that it is
not only the mere presence or position of the gene that is causing the
differences in phenotype.

[0908]21.3 Parameters Measured

Biomass-Related Parameter Measurement

[0909]From the stage of sowing until the stage of maturity the plants were
passed several times through a digital imaging cabinet. At each time
point digital images (2048×1536 pixels, 16 million colours) were
taken of each plant from at least 6 different angles.

[0910]The plant aboveground area (or leafy biomass) was determined by
counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This value was
averaged for the pictures taken on the same time point from the different
angles and was converted to a physical surface value expressed in square
mm by calibration. Experiments show that the aboveground plant area
measured this way correlates with the biomass of plant parts above
ground. The above ground area is the time point at which the plant had
reached its maximal leafy biomass. The early vigour is the plant
(seedling) aboveground area three weeks post-germination.

Seed-Related Parameter Measurements

[0911]The mature primary panicles were harvested, counted, bagged,
barcode-labeled and then dried for three days in an oven at 37° C.
The panicles were then threshed and all the seeds were collected and
counted. The filled husks were separated from the empty ones using an
air-blowing device. The empty husks were discarded and the remaining
fraction was counted again. The filled husks were weighed on an
analytical balance. The number of filled seeds was determined by counting
the number of filled husks that remained after the separation step. The
total seed yield was measured by weighing all filled husks harvested from
a plant. Total seed number per plant was measured by counting the number
of husks harvested from a plant. Thousand kernel weight (TKW) is
extrapolated from the number of filled seeds counted and their total
weight. The harvest index (HI) in the present invention is defined as the
ratio between the total seed yield and the above ground area (mm2),
multiplied by a factor 106. The total number of flowers per panicle
as defined in the present invention is the ratio between the total number
of seeds and the number of mature primary panicles. The seed fill rate as
defined in the present invention is the proportion (expressed as a %) of
the number of filled seeds over the total number of seeds (or florets).

Example 22

Results of the Phenotypic Evaluation of the Transgenic Plants

[0912]The results of the evaluation of transgenic rice plants expressing
the nucleic acid sequence useful in performing the methods of the
invention are presented in Table G. The percentage difference between the
transgenics and the corresponding nullizygotes is also shown, with a P
value from the F test below 0.05.

[0913]Total seed yield, number of filled seeds, seed fill rate and harvest
index are significantly increased in the transgenic plants expressing the
nucleic acid sequence useful in performing the methods of the invention,
compared to the control plants (in this case, the nullizygotes).

[0914]The rice SIK gene was amplified by PCR with primers (SEQ ID NO: 227;
sense, start codon in bold, AttB1 site in italic:
5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatgatgggttgcttcactgtc-3') and (SEQ
ID NO: 228; reverse, complementary, AttB2 site in italic:
5'-ggggaccactttgtacaagaaagctgggtatggacaatcaaaaaccctca-3'), which include
the AttB sites for Gateway recombination, were used for PCR
amplification. PCR was performed using Hifi Taq DNA polymerase under
standard conditions. The PCR fragment was purified using standard
methods. The first step of the Gateway procedure, the BP reaction, was
then performed, during which the PCR fragment recombines in vivo with the
pDONR201 plasmid to produce, according to the Gateway terminology, an
"entry clone". Plasmid pDONR201 was purchased from Invitrogen, as part of
the Gateway® technology.

Example 24

Vector Construction Downregulation

[0915]The entry clone was subsequently used in an LR reaction with a
destination vector used for Oryza sativa transformation for the
downregulation construct. This vectors contained within the T-DNA
borders: a plant selectable marker; a screenable marker expression
cassette; and a Gateway cassette intended for LR in vivo recombination so
as to integrate the sequence of interest from the entry clone in sense or
anti sense orientation. A rice GOS2 promoter (SEQ ID NO: 226) for
constitutive expression was located upstream of this Gateway cassette.

[0916]After the LR recombination step, the resulting expression vector
(FIG. 16) was transformed into Agrobacterium strain LBA4044 and
subsequently to Oryza sativa plants. Transformed rice plants were allowed
to grow and were then examined for the parameters described in Example
26.

Example 25

Vector Construction Overexpression

[0917]The entry clone was subsequently used in an LR reaction with a
destination vector used for plant (Oryza sativa) transformation. This
vector contained within the T-DNA borders: a plant selectable marker; a
screenable marker expression cassette; and a Gateway cassette intended
for LR in vivo recombination with the sequence of interest already cloned
in the entry clone. A rice GOS2 promoter for constitutive expression was
located upstream of this Gateway cassette.

[0918]After the LR recombination step, the resulting expression vector
(FIG. 17) was transformed into Agrobacterium strain LBA4044. Transformed
colonies were grown on YEP media and selected by respective antibiotics
for two days at 28° C. These Agrobacterium cultures were used for
the plant transformation. Transformed rice plants were allowed to grow
and were then examined for the parameters described in Example 26.

Example 26

Evaluation of Plants Transformed with SIK in Anti Sense Orientation

[0919]Approximately 15 to 20 independent T0 rice transformants were
generated. The primary transformants were transferred from a tissue
culture chamber to a greenhouse for growing and harvest of T1 seed. Six
events for which the T1 progeny segregated 3:1 for presence/absence of
the transgene, were retained. For each of these events, approximately 10
T1 seedlings containing the transgene (hetero- and homozygotes) and
approximately 10 T1 seedlings lacking the transgene (nullizygotes) were
selected by monitoring visual marker expression.

[0920]The selected T1 plants were transferred to a greenhouse. Each plant
received a unique barcode label to link unambiguously the phenotyping
data to the corresponding plant. The selected T1 plants were grown on
soil in 10 cm diameter pots under the following environmental settings:
photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime
temperature=28° C., night time temperature=22° C., relative
humidity=60-70%. Plants were grown under optimal watering conditions
until they approached the heading stage when irrigation was withheld.
Humidity probes were inserted in randomly chosen pots to monitor the soil
water content (SWC). When SWC dropped below 20%, the plants were
automatically re-watered to achieve optimal watering conditions again.
Transgenic plants and the corresponding nullizygotes were grown
side-by-side at random positions. From the stage of sowing until the
stage of maturity the plants were passed several times through a digital
imaging cabinet. At each time point digital images (2048×1536
pixels, 16 million colours) were taken of each plant from at least 6
different angles.

Drought Screen

[0921]Plants from T2 seeds are grown in potting soil under normal
conditions until they approached the heading stage. They are then
transferred to a "dry" section where irrigation is withheld. Humidity
probes are inserted in randomly chosen pots to monitor the soil water
content (SWC). When SWC goes below certain thresholds, the plants are
automatically re-watered continuously until a normal level was reached
again. The plants are then re-transferred again to normal conditions. The
rest of the cultivation (plant maturation, seed harvest) is the same as
for plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

Nitrogen use Efficiency Screen

[0922]Rice plants from T2 seeds are grown in potting soil under normal
conditions except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

[0923]The plant aboveground area (or leafy biomass) was determined by
counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This value was
averaged for the pictures taken on the same time point from the different
angles and was converted to a physical surface value expressed in square
mm by calibration. Experiments show that the aboveground plant area
measured this way correlates with the biomass of plant parts aboveground.
The Areamax is the aboveground area at the time point at which the plant
had reached its maximal leafy biomass.

[0924]The mature primary panicles were harvested, bagged, barcode-labelled
and then dried for three days in the oven at 37° C. The panicles
were then threshed and all the seeds collected. The filled husks were
separated from the empty ones using an air-blowing device. After
separation, both seed lots were then counted using a commercially
available counting machine. The empty husks were discarded. The filled
husks were weighed on an analytical balance and the cross-sectional area
of the seeds was measured using digital imaging. This procedure resulted
in the set of the following seed-related parameters:

[0925]The flowers-per-panicle is a parameter estimating the average number
of florets per panicle on a plant, derived from the number of total seeds
divided by the number of first panicles. The tallest panicle and all the
panicles that overlapped with the tallest panicle when aligned
vertically, were considered as first panicles and were counted manually.
The number of filled seeds was determined by counting the number of
filled husks that remained after the separation step. The total seed
yield (total seed weight) was measured by weighing all filled husks
harvested from a plant. Total seed number per plant was measured by
counting the number of husks harvested from a plant and corresponds to
the number of florets per plant. These parameters were derived in an
automated way from the digital images using image analysis software and
were analysed statistically. Individual seed parameters (including width,
length, area, weight) were measured using a custom-made device consisting
of two main components, a weighing and imaging device, coupled to
software for image analysis. The harvest index in the present invention
is defined as the ratio between the total seed yield (g) and the above
ground area (mm2), multiplied by a factor 106.

[0926]A two factor ANOVA (analyses of variance) corrected for the
unbalanced design was used as statistical model for the overall
evaluation of plant phenotypic characteristics. An F-test was carried out
on all the parameters measured of all the plants of all the events
transformed with that gene. The F-test was carried out to check for an
effect of the gene over all the transformation events and to verify for
an overall effect of the gene, also named herein "global gene effect". If
the value of the F test shows that the data are significant, than it is
concluded that there is a "gene" effect, meaning that not only presence
or the position of the gene is causing the effect. The threshold for
significance for a true global gene effect is set at 5% probability level
for the F test.

[0927]To check for an effect of the genes within an event, i.e., for a
line-specific effect, a t-test was performed within each event using data
sets from the transgenic plants and the corresponding null plants. "Null
plants" or "null segregants" or "nullizygotes" are the plants treated in
the same way as the transgenic plant, but from which the transgene has
segregated. Null plants can also be described as the homozygous negative
transformed plants. The threshold for significance for the t-test is set
at 10% probability level. The results for some events can be above or
below this threshold. This is based on the hypothesis that a gene might
only have an effect in certain positions in the genome, and that the
occurrence of this position-dependent effect is not uncommon. This kind
of gene effect is also named herein a "line effect of the gene". The
p-value was obtained by comparing the t-value to the t-distribution or
alternatively, by comparing the F-value to the F-distribution. The
p-value then gives the probability that the null hypothesis (i.e., that
there is no effect of the transgene) is correct.

Example 27

Results

1. Thousand Kernel Weight

[0928]The transgenic lines transformed with the downregulation construct
gave an overall percentage increase of 3% compared to controls with the
best line giving a 10% increase compared to controls.

2. Harvest Index

[0929]The transgenic lines transformed with the downregulation construct
gave an overall percentage increase of 16% compared to controls with the
best line giving a 61% increase compared to controls.

3. Fill Rate

[0930]The transgenic lines transformed with the downregulation construct
gave an overall percentage increase of 14% compared to controls with the
best line giving a 41% increase compared to controls.

4. Flowers per Panicle (Number of Flowers per Plant)

[0931]The transgenic lines transformed with the downregulation construct
gave an overall percentage decrease of -3% compared to controls with the
best line giving a -11% decrease compared to controls. A decrease in the
number of flowers may be important for grasses (when used for lawns for
example).

[0932]The transgenic lines transformed with the overexpression construct
gave an overall percentage increase of 6% compared to controls with the
best line giving a 14% increase compared to controls.

[0933]Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO:
229 and SEQ ID NO: 230 were identified amongst those maintained in the
Entrez Nucleotides database at the National Center for Biotechnology
Information (NCBI) using database sequence search tools, such as the
Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.
215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
This program was used to find regions of local similarity between
sequences by comparing nucleic acid or polypeptide sequences to sequence
databases and by calculating the statistical significance of matches. The
polypeptide encoded by SEQ ID NO: 229 was used for the TBLASTN algorithm,
with default settings and with the filter to ignore low complexity
sequences set off. The output of the analysis was viewed by pairwise
comparison, and ranked according to the probability score (E-value),
where the score reflects the probability that a particular alignment
occurs by chance (the lower the E-value, the more significant the hit).
In addition to E-values, comparisons were also scored by percentage
identity. Percentage identity refers to the number of identical
nucleotides (or amino acids) between the two compared nucleic acid (or
polypeptide) sequences over a particular length.

[0935]AlignX from the Vector NTI (Invitrogen) is based on the popular
Clustal algorithm of progressive alignment (Thompson et al. (1997)
Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res
31:3497-3500). A phylogenetic tree was constructed using a
neighbour-joining clustering algorithm (see FIG. 18). Default values are
for the gap open penalty of 10, for the gap extension penalty of 0.1 and
the selected weight matrix is Blosum 62 (if polypeptides are aligned).
The result of the multiple sequence alignment using is shown in FIG. 19.

Example 30

Identification of Domains Comprised in Polypeptide Sequences Useful in
Performing the Methods of the Invention

[0936]The Integrated Resource of Protein Families, Domains and Sites
(InterPro) database is an integrated interface for the commonly used
signature databases for text- and sequence-based searches. The InterPro
database combines these databases, which use different methodologies and
varying degrees of biological information about well-characterized
proteins to derive protein signatures. Collaborating databases include
SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs.
Interpro is hosted at the European Bioinformatics Institute in the United
Kingdom.

[0937]The results of the InterPro scan of the polypeptide sequence as
represented by SEQ ID NO: 230 are presented in Table I.

[0938]The Arabidopsis thaliana HAT4-encoding gene was amplified by PCR
using as a template an Arabidopsis thaliana cDNA library (Invitrogen,
Paisley, UK). After reverse transcription of RNA extracted from
seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size
of the bank was 1.6 kb and the original number of clones was of the order
of 1.67×107 cfu. Original titer was determined to be
3.34×106 cfu/ml after first amplification of 6×1010
cfu/ml. After plasmid extraction, 200 ng of template was used in a 50
μl PCR mix. The primers used were:

which include the AttB sites for Gateway recombination. The amplified PCR
fragment was purified also using standard methods. The first step of the
Gateway procedure, the BP reaction, was then performed, during which the
PCR fragment recombined in vivo with the pDONR201 plasmid to produce,
according to the Gateway terminology, an "entry clone". Plasmid pDONR201
was purchased from Invitrogen, as part of the Gateway® technology.

[0939]The entry clone was subsequently used in an LR reaction with a
destination vector used for Oryza sativa transformation. This vector
contains as functional elements within the T-DNA borders: a plant
selectable marker; a screenable marker expression cassette; and a Gateway
cassette intended for LR in vivo recombination with the nucleic acid
sequence of interest already cloned in the entry clone. A rice GOS2
promoter (SEQ ID NO: 282) for constitutive expression was located
upstream of this Gateway cassette.

[0940]After the LR recombination step, the resulting expression vector
(FIG. 21) was transformed into Agrobacterium strain LBA4044 according to
methods well known in the art.

Example 33

Plant Transformation

Rice Transformation

[0941]The Agrobacterium containing the expression vector was used to
transform Oryza sativa plants. Mature dry seeds of the rice japonica
cultivar Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl2, followed by a 6 times 15 minutes wash with sterile distilled
water. The sterile seeds were then germinated on a medium containing
2,4-D (callus induction medium). After incubation in the dark for four
weeks, embryogenic, scutellum-derived calli were excised and propagated
on the same medium. After two weeks, the calli were multiplied or
propagated by subculture on the same medium for another 2 weeks.
Embryogenic callus pieces were sub-cultured on fresh medium 3 days before
co-cultivation (to boost cell division activity).

[0942]Agrobacterium strain LBA4404 containing the expression vector was
used for cocultivation. Agrobacterium was inoculated on AB medium with
the appropriate antibiotics and cultured for 3 days at 28° C. The
bacteria were then collected and suspended in liquid co-cultivation
medium to a density (OD600) of about 1. The suspension was then
transferred to a Petri dish and the calli immersed in the suspension for
15 minutes. The callus tissues were then blotted dry on a filter paper
and transferred to solidified, co-cultivation medium and incubated for 3
days in the dark at 25° C. Co-cultivated calli were grown on
2,4-D-containing medium for 4 weeks in the dark at 28° C. in the
presence of a selection agent. During this period, rapidly growing
resistant callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to five
weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks
on an auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse.

[0943]Approximately 35 independent T0 rice transformants were generated
for one construct. The primary transformants were transferred from a
tissue culture chamber to a greenhouse. After a quantitative PCR analysis
to verify copy number of the T-DNA insert, only single copy transgenic
plants that exhibit tolerance to the selection agent were kept for
harvest of T1 seed. Seeds were then harvested three to five months after
transplanting. The method yielded single locus transformants at a rate of
over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).

Example 34

Phenotypic Evaluation Procedure

34.1 Evaluation Setup

[0944]Approximately 35 independent T0 rice transformants were generated.
The primary transformants were transferred from a tissue culture chamber
to a greenhouse for growing and harvest of T1 seed. Eight events, of
which the T1 progeny segregated 3:1 for presence/absence of the
transgene, were retained. For each of these events, approximately 10 T1
seedlings containing the transgene (hetero- and homo-zygotes) and
approximately 10 T1 seedlings lacking the transgene (nullizygotes) were
selected by monitoring visual marker expression. The transgenic plants
and the corresponding nullizygotes were grown side-by-side at random
positions. Greenhouse conditions were of shorts days (12 hours light),
28° C. in the light and 22° C. in the dark, and a relative
humidity of 70%.

[0945]Four T1 events were further evaluated in the T2 generation following
the same evaluation procedure as for the T1 generation but with more
individuals per event.

Drought Screen

[0946]Plants from T2 seeds are grown in potting soil under normal
conditions until they approached the heading stage. They are then
transferred to a "dry" section where irrigation is withheld. Humidity
probes are inserted in randomly chosen pots to monitor the soil water
content (SWC). When SWC goes below certain thresholds, the plants are
automatically re-watered continuously until a normal level was reached
again. The plants are then re-transferred again to normal conditions. The
rest of the cultivation (plant maturation, seed harvest) is the same as
for plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

Nitrogen use Efficiency Screen

[0947]Rice plants from T2 seeds are grown in potting soil under normal
conditions except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

[0948]Non-destructive oil measurements from rice seeds was measured using
the Oxford QP20+ pulsed NMR. Whole seeds were used without dehusking.

34.2 Statistical Analysis: F-Test

[0949]A two factor ANOVA (analysis of variants) was used as a statistical
model for the overall evaluation of plant phenotypic characteristics. An
F-test was carried out on all the parameters measured of all the plants
of all the events transformed with the gene of the present invention. The
F-test was carried out to check for an effect of the gene over all the
transformation events and to verify an overall effect of the gene, also
known as a global gene effect. The threshold for significance for a true
global gene effect was set at a 5% probability level for the F-test. A
significant F-test value points to a gene effect, meaning that it is not
only the mere presence or position of the gene that is causing the
differences in phenotype.

34.3 Results

[0950]The best line showed an 11% increase in oil content compared to
control plants, with an average increase in oil content of 6% over all
lines and a p value from the F-test of <0.0001

Example 35

Transformation of Corn

[0951]Transformation of maize (Zea mays) is performed with a modification
of the method described by Ishida et al. (1996) Nature Biotech 14(6):
745-50. Transformation is genotype-dependent in corn and only specific
genotypes are amenable to transformation and regeneration. The inbred
line A188 (University of Minnesota) or hybrids with A188 as a parent are
good sources of donor material for tansformation, but other genotypes can
be used successfully as well. Ears are harvested from corn plant
approximately 11 days after pollination (DAP) when the length of the
immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated
with Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised embryos
are grown on callus induction medium, then maize regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to maize rooting medium and
incubated at 25° C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Example 36

Transformation of Wheat

[0952]Transformation of wheat is performed with the method described by
Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite
(available from CIMMYT, Mexico) is commonly used in transformation.
Immature embryos are co-cultivated with Agrobacterium tumefaciens
containing the expression vector, and transgenic plants are recovered
through organogenesis. After incubation with Agrobacterium, the embryos
are grown in vitro on callus induction medium, then regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and incubated
at 25° C. for 2-3 weeks, until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Example 37

Transformation of Soybean

[0953]Soybean is transformed according to a modification of the method
described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several
commercial soybean varieties are amenable to transformation by this
method. The cultivar Jack (available from the Illinois Seed foundation)
is commonly used for transformation. Soybean seeds are sterilised for in
vitro sowing. The hypocotyl, the radicle and one cotyledon are excised
from seven-day old young seedlings. The epicotyl and the remaining
cotyledon are further grown to develop axillary nodes. These axillary
nodes are excised and incubated with Agrobacterium tumefaciens containing
the expression vector. After the cocultivation treatment, the explants
are washed and transferred to selection media. Regenerated shoots are
excised and placed on a shoot elongation medium. Shoots no longer than 1
cm are placed on rooting medium until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Example 38

Transformation of Rapeseed/Canola

[0954]Cotyledonary petioles and hypocotyls of 5-6 day old young seedling
are used as explants for tissue culture and transformed according to
Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar
Westar (Agriculture Canada) is the standard variety used for
transformation, but other varieties can also be used. Canola seeds are
surface-sterilized for in vitro sowing. The cotyledon petiole explants
with the cotyledon attached are excised from the in vitro seedlings, and
inoculated with Agrobacterium (containing the expression vector) by
dipping the cut end of the petiole explant into the bacterial suspension.
The explants are then cultured for 2 days on MSBAP-3 medium containing 3
mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on
MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection
agent until shoot regeneration. When the shoots are 5-10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred
to the rooting medium (MSO) for root induction. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from plants
that exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.

Example 39

Transformation of Alfalfa

[0955]A regenerating clone of alfalfa (Medicago sativa) is transformed
using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847).
Regeneration and transformation of alfalfa is genotype dependent and
therefore a regenerating plant is required. Methods to obtain
regenerating plants have been described. For example, these can be
selected from the cultivar Rangelander (Agriculture Canada) or any other
commercial alfalfa variety as described by Brown DCW and A Atanassov
(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the
RA3 variety (University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants
are cocultivated with an overnight culture of Agrobacterium tumefaciens
C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing the expression vector. The explants are cocultivated for 3 d
in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and Skoog,
1962) and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to inhibit
Agrobacterium growth. After several weeks, somatic embryos are
transferred to BOi2Y development medium containing no growth regulators,
no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings were
transplanted into pots and grown in a greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

[0956]Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO:
285 and/or protein sequences related to SEQ ID NO: 286 were identified
amongst those maintained in the Entrez Nucleotides database at the
National Center for Biotechnology Information (NCBI) using database
sequence search tools, such as the Basic Local Alignment Tool (BLAST)
(Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al.
(1997) Nucleic Acids Res. 25:3389-3402). The program is used to find
regions of local similarity between sequences by comparing nucleic acid
or polypeptide sequences to sequence databases and by calculating the
statistical significance of matches. The polypeptide encoded by SEQ ID
NO: 285 was used for the TBLASTN algorithm, with default settings and the
filter to ignore low complexity sequences set off. The output of the
analysis was viewed by pairwise comparison, and ranked according to the
probability score (E-value), where the score reflects the probability
that a particular alignment occurs by chance (the lower the E-value, the
more significant the hit). In addition to E-values, comparisons were also
scored by percentage identity. Percentage identity refers to the number
of identical nucleotides (or amino acids) between the two compared
nucleic acid (or polypeptide) sequences over a particular length. In some
instances, the default parameters may be adjusted to modify the
stringency of the search.

[0957]In addition to the publicly available nucleic acid sequences
available at NCBI, proprietary sequence databases are also searched
following the same procedure as described herein above.

[0959]AlignX from the Vector NTI (Invitrogen) is based on the popular
Clustal algorithm of progressive alignment (Thompson et al. (1997)
Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res
31:3497-3500). A phylogenetic tree can be constructed using a
neighbour-joining clustering algorithm. Default values are for the gap
open penalty of 10, for the gap extension penalty of 0.1 and the selected
weight matrix is Blosum 62 (if polypeptides are aligned).

[0960]The result of a multiple sequence alignment using polypeptides
relevant in identifying the ones useful in performing the methods of the
invention is shown in FIG. 23a of the present application. The three
Zinc-finger domains can be easily identified.

Example 42

Calculation of Global Percentage Identity between Polypeptide Sequences
useful in Performing the Methods of the Invention

[0961]Global percentages of similarity and identity between full length
polypeptide sequences useful in performing the methods of the invention
were determined using one of the methods available in the art, the MatGAT
(Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29.
MatGAT: an application that generates similarity/identity matrices using
protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software
hosted by Ledion Bitincka). MatGAT software generates similarity/identity
matrices for DNA or protein sequences without needing pre-alignment of
the data. The program performs a series of pair-wise alignments using the
Myers and Miller global alignment algorithm (with a gap opening penalty
of 12, and a gap extension penalty of 2), calculates similarity and
identity using for example Blosum 62 (for polypeptides), and then places
the results in a distance matrix. Sequence similarity is shown in the
bottom half of the dividing line and sequence identity is shown in the
top half of the diagonal dividing line.

[0965]Results of the software analysis are shown in Table K for the global
similarity and identity over the full length of the polypeptide sequences
(excluding the partial polypeptide sequences). Percentage identity is
given above the diagonal and percentage similarity is given below the
diagonal.

[0966]The percentage identity between the polypeptide sequences useful in
performing the methods of the invention can be as low as 16.9% amino acid
identity compared to SEQ ID NO: 286.

Identification of Domains Comprised in Polypeptide Sequences useful in
Performing the Methods of the Invention

[0967]The Integrated Resource of Protein Families, Domains and Sites
(InterPro) database is an integrated interface for the commonly used
signature databases for text- and sequence-based searches. The InterPro
database combines these databases, which use different methodologies and
varying degrees of biological information about well-characterized
proteins to derive protein signatures. Collaborating databases include
SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs.
Interpro is hosted at the European Bioinformatics Institute in the United
Kingdom.

[0968]The results of the InterPro scan of the polypeptide sequence as
represented by SEQ ID NO: 286 are presented in Table L.

[0969]TargetP 1.1 predicts the subcellular location of eukaryotic
proteins. The location assignment is based on the predicted presence of
any of the N-terminal pre-sequences: chloroplast transit peptide (cTP),
mitochondrial targeting peptide (mTP) or secretory pathway signal peptide
(SP). Scores on which the final prediction is based are not really
probabilities, and they do not necessarily add to one. However, the
location with the highest score is the most likely according to TargetP,
and the relationship between the scores (the reliability class) may be an
indication of how certain the prediction is. The reliability class (RC)
ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP
is maintained at the server of the Technical University of Denmark.

[0970]For the sequences predicted to contain an N-terminal presequence a
potential cleavage site can also be predicted.

[0971]A number of parameters were selected, such as organism group
(non-plant or plant), cutoff sets (none, predefined set of cutoffs, or
user-specified set of cutoffs), and the calculation of prediction of
cleavage sites (yes or no).

[0972]The results of TargetP 1.1 analysis of the polypeptide sequence as
represented by SEQ ID NO: 286 are presented Table M. The "plant" organism
group has been selected, no cutoffs defined, and the predicted length of
the transit peptide requested. There is no clear prediction of the
subcellular localisation, only a weak prediction for a mitochondrial
localisation (reliability class 5, which is the lowest reliability). SYB1
proteins therefore may also be located in the cytoplasm.

[0973]Many other algorithms can be used to perform such analyses,
including: [0974]ChloroP 1.1 hosted on the server of the Technical
University of Denmark; [0975]Protein Prowler Subcellular Localisation
Predictor version 1.2 hosted on the server of the Institute for Molecular
Bioscience, University of Queensland, Brisbane, Australia; [0976]PENCE
Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of
Alberta, Edmonton, Alberta, Canada;

Example 45

Assay Related to the Polypeptide Sequences Useful in Performing the
Methods of the Invention

[0977]The polypeptide sequence as represented by SEQ ID NO: 286 may
interact with nucleic acids as well as with proteins, by virtue of the
presence of the Zinc finger domains. DNA binding assays are well known in
the art, including PCR-assisted DNA binding site selection and a DNA
binding gel-shift assay; for a general reference, see Current Protocols
in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current
Protocols.

[0978]The protein represented by SEQ ID NO: 286 is predicted to interact
with several proteins (results from analysis with the SMART database), as
shown in Table N. The functionality of a SYB1 protein may thus be tested
in a yeast two-hybrid screen with candidate ligand proteins.

[0979]Furthermore, expression of a SYB1 protein according to the methods
of the present invention results in increased seed yield as described
below.

Example 46

Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO: 285

[0980]Unless otherwise stated, recombinant DNA techniques are performed
according to standard protocols described in (Sambrook (2001) Molecular
Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory
Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994),
Current Protocols in Molecular Biology, Current Protocols. Standard
materials and methods for plant molecular work are described in Plant
Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific Publications
(UK).

[0981]The Arabidopsis thaliana SYB1 gene was amplified by PCR using as
template an Arabidopsis thaliana seedling cDNA library (Invitrogen,
Paisley, UK). After reverse transcription of RNA extracted from
seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size
of the bank was 1.5 kb and the original number of clones was of the order
of 1.59×107 cfu. Original titer was determined to be 9.6×105
cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid
extraction, 200 ng of template was used in a 50 μl PCR mix. Primers
prm5539 (SEQ ID NO: 341; sense, start codon in bold, AttB1 site in
italic: 5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatgagcagacccggagatt -3')
and prm5540 (SEQ ID NO: 342; reverse, complementary, Att B2 site in
italic: 5'-ggggaccactttgtacaagaaagctgggtagacaaggctacttcaaaagca -3'),
which include the AttB sites for Gateway recombination, were used for PCR
amplification. PCR was performed using Hifi Taq DNA polymerase in
standard conditions. A PCR fragment of 559 by (including attB sites) was
amplified and purified also using standard methods. The first step of the
Gateway procedure, the BP reaction, was then performed, during which the
PCR fragment recombines in vivo with the pDONR201 plasmid to produce,
according to the Gateway terminology, an "entry clone", p58a. Plasmid
pDONR201 was purchased from Invitrogen, as part of the Gateway®
technology.

[0982]The entry clone p58a was subsequently used in an LR reaction with
p0640, a destination vector used for Oryza sativa transformation. This
vector contains as functional elements within the T-DNA borders: a plant
selectable marker; a screenable marker expression cassette; and a Gateway
cassette intended for LR in vivo recombination with the nucleic acid
sequence of interest already cloned in the entry clone. A rice GOS2
promoter (SEQ ID NO: 343) for constitutive expression (pGOS2) was located
upstream of this Gateway cassette.

[0983]After the LR recombination step, the resulting expression vector
pGOS2::SYB1 (FIG. 24) was transformed into Agrobacterium strain LBA4044
according to methods well known in the art.

Example 48

Plant Transformation

Rice Transformation

[0984]The Agrobacterium containing the expression vector was used to
transform Oryza sativa plants. Mature dry seeds of the rice japonica
cultivar Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl2, followed by a 6 times 15 minutes wash with sterile distilled
water. The sterile seeds were then germinated on a medium containing
2,4-D (callus induction medium). After incubation in the dark for four
weeks, embryogenic, scutellum-derived calli were excised and propagated
on the same medium. After two weeks, the calli were multiplied or
propagated by subculture on the same medium for another 2 weeks.
Embryogenic callus pieces were sub-cultured on fresh medium 3 days before
co-cultivation (to boost cell division activity).

[0985]Agrobacterium strain LBA4404 containing the expression vector was
used for cocultivation. Agrobacterium was inoculated on AB medium with
the appropriate antibiotics and cultured for 3 days at 28° C. The
bacteria were then collected and suspended in liquid co-cultivation
medium to a density (OD600) of about 1. The suspension was then
transferred to a Petri dish and the calli immersed in the suspension for
15 minutes. The callus tissues were then blotted dry on a filter paper
and transferred to solidified, co-cultivation medium and incubated for 3
days in the dark at 25° C. Co-cultivated calli were grown on
2,4-D-containing medium for 4 weeks in the dark at 28° C. in the
presence of a selection agent. During this period, rapidly growing
resistant callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to five
weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks
on an auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse.

[0986]Approximately 35 independent T0 rice transformants were generated
for one construct. The primary transformants were transferred from a
tissue culture chamber to a greenhouse. After a quantitative PCR analysis
to verify copy number of the T-DNA insert, only single copy transgenic
plants that exhibit tolerance to the selection agent were kept for
harvest of T1 seed. Seeds were then harvested three to five months after
transplanting. The method yielded single locus transformants at a rate of
over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

[0987]Transformation of maize (Zea mays) is performed with a modification
of the method described by Ishida et al. (1996) Nature Biotech 14(6):
745-50. Transformation is genotype-dependent in corn and only specific
genotypes are amenable to transformation and regeneration. The inbred
line A188 (University of Minnesota) or hybrids with A188 as a parent are
good sources of donor material for transformation, but other genotypes
can be used successfully as well. Ears are harvested from corn plant
approximately 11 days after pollination (DAP) when the length of the
immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated
with Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised embryos
are grown on callus induction medium, then maize regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to maize rooting medium and
incubated at 25° C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Wheat Transformation

[0988]Transformation of wheat is performed with the method described by
Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite
(available from CIMMYT, Mexico) is commonly used in transformation.
Immature embryos are co-cultivated with Agrobacterium tumefaciens
containing the expression vector, and transgenic plants are recovered
through organogenesis. After incubation with Agrobacterium, the embryos
are grown in vitro on callus induction medium, then regeneration medium,
containing the selection agent (for example imidazolinone but various
selection markers can be used). The Petri plates are incubated in the
light at 25° C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and incubated
at 25° C. for 2-3 weeks, until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Soybean Transformation

[0989]Soybean is transformed according to a modification of the method
described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several
commercial soybean varieties are amenable to transformation by this
method. The cultivar Jack (available from the Illinois Seed foundation)
is commonly used for transformation. Soybean seeds are sterilised for in
vitro sowing. The hypocotyl, the radicle and one cotyledon are excised
from seven-day old young seedlings. The epicotyl and the remaining
cotyledon are further grown to develop axillary nodes. These axillary
nodes are excised and incubated with Agrobacterium tumefaciens containing
the expression vector. After the cocultivation treatment, the explants
are washed and transferred to selection media. Regenerated shoots are
excised and placed on a shoot elongation medium. Shoots no longer than 1
cm are placed on rooting medium until roots develop. The rooted shoots
are transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that contain a
single copy of the T-DNA insert.

Rapeseed/Canola Transformation

[0990]Cotyledonary petioles and hypocotyls of 5-6 day old young seedling
are used as explants for tissue culture and transformed according to
Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar
Westar (Agriculture Canada) is the standard variety used for
transformation, but other varieties can also be used. Canola seeds are
surface-sterilized for in vitro sowing. The cotyledon petiole explants
with the cotyledon attached are excised from the in vitro seedlings, and
inoculated with Agrobacterium (containing the expression vector) by
dipping the cut end of the petiole explant into the bacterial suspension.
The explants are then cultured for 2 days on MSBAP-3 medium containing 3
mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on
MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection
agent until shoot regeneration. When the shoots are 5-10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred
to the rooting medium (MS0) for root induction. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from plants
that exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.

Alfalfa Transformation

[0991]A regenerating clone of alfalfa (Medicago sativa) is transformed
using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847).
Regeneration and transformation of alfalfa is genotype dependent and
therefore a regenerating plant is required. Methods to obtain
regenerating plants have been described. For example, these can be
selected from the cultivar Rangelander (Agriculture Canada) or any other
commercial alfalfa variety as described by Brown D C W and A Atanassov
(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the
RA3 variety (University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are
cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1
pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing the expression vector. The explants are cocultivated for 3 d
in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and Skoog,
1962) and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to inhibit
Agrobacterium growth. After several weeks, somatic embryos are
transferred to BOi2Y development medium containing no growth regulators,
no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings were
transplanted into pots and grown in a greenhouse. T1 seeds are produced
from plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.

Example 49

Phenotypic Evaluation Procedure

49.1 Evaluation Setup

[0992]Approximately 35 independent T0 rice transformants were generated.
The primary transformants were transferred from a tissue culture chamber
to a greenhouse for growing and harvest of T1 seed. Six events, of which
the T1 progeny segregated 3:1 for presence/absence of the transgene, were
retained. For each of these events, approximately 10 T1 seedlings
containing the transgene (hetero- and homo-zygotes) and approximately 10
T1 seedlings lacking the transgene (nullizygotes) were selected by
monitoring visual marker expression. The transgenic plants and the
corresponding nullizygotes were grown side-by-side at random positions.
Greenhouse conditions were of shorts days (12 hours light), 28° C.
in the light and 22° C. in the dark, and a relative humidity of
70%.

Drought Screen

[0993]Plants from T2 seeds are grown in potting soil under normal
conditions until they approached the heading stage. They are then
transferred to a "dry" section where irrigation is withheld. Humidity
probes are inserted in randomly chosen pots to monitor the soil water
content (SWC). When SWC goes below certain thresholds, the plants are
automatically re-watered continuously until a normal level was reached
again. The plants are then re-transferred again to normal conditions. The
rest of the cultivation (plant maturation, seed harvest) is the same as
for plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

Nitrogen use Efficiency Screen

[0994]Rice plants from T2 seeds are grown in potting soil under normal
conditions except for the nutrient solution. The pots are watered from
transplantation to maturation with a specific nutrient solution
containing reduced N nitrogen (N) content, usually between 7 to 8 times
less. The rest of the cultivation (plant maturation, seed harvest) is the
same as for plants not grown under abiotic stress. Growth and yield
parameters are recorded as detailed for growth under normal conditions.

[0995]Four T1 events were further evaluated in the T2 generation following
the same evaluation procedure as for the T1 generation but with more
individuals per event. From the stage of sowing until the stage of
maturity the plants were passed several times through a digital imaging
cabinet. At each time point digital images (2048×1536 pixels, 16
million colours) were taken of each plant from at least 6 different
angles.

49.2 Statistical Analysis: F-Test

[0996]A two factor ANOVA (analysis of variants) was used as a statistical
model for the overall evaluation of plant phenotypic characteristics. An
F-test was carried out on all the parameters measured of all the plants
of all the events transformed with the gene of the present invention. The
F-test was carried out to check for an effect of the gene over all the
transformation events and to verify for an overall effect of the gene,
also known as a global gene effect. The threshold for significance for a
true global gene effect was set at a 5% probability level for the F-test.
A significant F-test value points to a gene effect, meaning that it is
not only the mere presence or position of the gene that is causing the
differences in phenotype.

49.3 Parameters Measured

Biomass-Related Parameter Measurement

[0997]From the stage of sowing until the stage of maturity the plants were
passed several times through a digital imaging cabinet. At each time
point digital images (2048×1536 pixels, 16 million colours) were
taken of each plant from at least 6 different angles.

[0998]The plant aboveground area (or leafy biomass) was determined by
counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This value was
averaged for the pictures taken on the same time point from the different
angles and was converted to a physical surface value expressed in square
mm by calibration. Experiments show that the aboveground plant area
measured this way correlates with the biomass of plant parts above
ground. The above ground area is the time point at which the plant had
reached its maximal leafy biomass. The early vigour is the plant
(seedling) aboveground area three weeks post-germination. Increase in
root biomass is expressed as an increase in total root biomass (measured
as maximum biomass of roots observed during the lifespan of a plant); or
as an increase in the root/shoot index (measured as the ratio between
root mass and shoot mass in the period of active growth of root and
shoot).

Seed-Related Parameter Measurements

[0999]The mature primary panicles were harvested, counted, bagged,
barcode-labelled and then dried for three days in an oven at 37°
C. The panicles were then threshed and all the seeds were collected and
counted. The filled husks were separated from the empty ones using an
air-blowing device. The empty husks were discarded and the remaining
fraction was counted again. The filled husks were weighed on an
analytical balance. The number of filled seeds was determined by counting
the number of filled husks that remained after the separation step. The
total seed yield was measured by weighing all filled husks harvested from
a plant. Total seed number per plant was measured by counting the number
of husks harvested from a plant. Thousand Kernel Weight (TKW) is
extrapolated from the number of filled seeds counted and their total
weight. The Harvest Index (HI) in the present invention is defined as the
ratio between the total seed yield and the above ground area (mm2),
multiplied by a factor 106. The total number of flowers per panicle as
defined in the present invention is the ratio between the total number of
seeds and the number of mature primary panicles. The seed fill rate as
defined in the present invention is the proportion (expressed as a %) of
the number of filled seeds over the total number of seeds (or florets).

Example 50

Results of the Phenotypic Evaluation of the Transgenic Plants

[1000]The results of the evaluation of transgenic rice plants expressing
the nucleic acid sequence useful in performing the methods of the
invention are presented in Table 0. The percentage difference between the
transgenics and the corresponding nullizygotes is also shown, with a P
value from the F test below 0.05.

[1001]Total seed yield, number of filled seeds, seed fill rate, harvest
index and thousand kernel weight are significantly increased in the
transgenic plants expressing the nucleic acid sequence useful in
performing the methods of the invention, compared to the control plants
(in this case, the nullizygotes).

Sequence CWU
0
SQTB
SEQUENCE LISTING
The patent application contains a lengthy "Sequence Listing" section. A
copy of the "Sequence Listing" is available in electronic form from the
USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100218271A1).
An electronic copy of the "Sequence Listing" will also be available from
the USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).